Method for manufacturing a shaped crosslinked hyaluronic acid product

ABSTRACT

A method for manufacturing a shaped cross-linked hyaluronic acid product, the method including the step of subjecting a non-cross-linked precipitated hyaluronic acid substrate in a desired shape to a single cross-linking reaction in a liquid medium having a pH of 11.5 or higher and including one or more polyfunctional cross-linking agent(s) and an amount of one or more organic solvent(s) giving precipitating conditions for hyaluronic acid, under suitable conditions to obtain a precipitated, shaped cross-linked hyaluronic acid product having a degree of modification of 1-40 cross-linking agent units per 1000 disaccharide units.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the field of polysaccharides. Morespecifically, the present invention is concerned with novel methods ofcross-linking hyaluronic acid and of manufacturing shaped cross-linkedhyaluronic acid products.

BACKGROUND OF THE INVENTION

One of the most widely used biocompatible polymers for medical use ishyaluronic acid. It is a naturally occurring polysaccharide belonging tothe group of glycosaminoglycans (GAGs). Hyaluronic acid and the otherGAGs are negatively charged heteropolysaccharide chains which have acapacity to absorb large amounts of water. Hyaluronic acid and productsderived from hyaluronic acid are widely used in the biomedical andcosmetic fields, for instance during viscosurgery and as a dermalfiller.

Water-absorbing gels, or hydrogels, are widely used in the biomedicalfield. They are generally prepared by chemical cross-linking of polymersto infinite networks. While native hyaluronic acid and certaincross-linked hyaluronic acid products absorb water until they arecompletely dissolved, cross-linked hyaluronic acid gels typically absorba certain amount of water until they are saturated, i.e. they have afinite liquid retention capacity, or swelling degree.

When preparing gels from biocompatible polymers, it is advantageous toensure a low degree of cross-linking so as to maintain a highbiocompatibility. However, often a more dense gel is required to have aproper biomedical effect, and in such a case the biocompatibility willoften be lost.

Since hyaluronic acid is present with identical chemical structureexcept for its molecular mass in most living organisms, it gives aminimum of reactions and allows for advanced medical uses. Cross-linkingand/or other modifications of the hyaluronic acid molecule is necessaryto improve its resistance to degradation or duration in vivo.Furthermore, such modifications affect the liquid retention capacity ofthe hyaluronic acid molecule. As a consequence thereof, hyaluronic acidhas been the subject of many modification attempts.

Since cross-linked hyaluronic acid gel products are highly complexchemical structures, they are typically characterised by a combinationof their chemical structures and their physical properties. Thedeviation in chemical structure from unmodified hyaluronic acid istypically reported as degree of modification, modification degree,cross-linking degree, cross-linking index or chemical modification,which all relate to the amount of cross-linking agent covalently boundto the hyaluronic acid. Throughout this text, the term degree ofmodification will be used. The most relevant physical properties of thecross-linked hyaluronic acid gel product are the volume of liquid thatthe gel can absorb and the rheological properties of the gel. Bothproperties describe the structural stability of the gel, often referredto as gel strength or firmness, but while the absorption of liquid canbe determined for a dry gel, the rheological properties have to bemeasured on a gel that is swollen to a desired concentration.Traditional expressions for the liquid absorption are swelling, swellingcapacity, liquid retention capacity, swelling degree, degree ofswelling, maximum liquid uptake and maximum swelling. Throughout thistext, the term swelling degree will be used. Regarding the rheologicalproperties of cross-linked hyaluronic acid gel products, it can be notedthat rotational rheometry is only useful for determining the rheology ofliquids, whereas oscillating rheometry is necessary to determine therheology of gels. The measurement yields the resistance of the gel todeformation in units of elastic modulus and viscous modulus. A high gelstrength will give a large resistance to deformation of the gel productswollen to a desired concentration.

US 2007/0066816 discloses a process for preparing double cross-linkedhyaluronic acid, involving cross-linking of a hyaluronic acid substratein two steps with an epoxide and a carbodiimide, respectively.

EP 2 199 308 A1 discloses cross-linking of a hyaluronan powder which isdispersed in a a liquid medium containing ethanol. The resultingproducts have a poorly controlled shape.

US 2012/0034462 A1 suggests without experimental evidence that thinstrands of cross-linked HA gel can be produced by passing a solid massof the cross-linked HA gel through a sieve or mesh.

Despite advances in the field, there remains a need for alternativemethods of manufacturing shaped cross-linked hyaluronic acid productshaving suitable liquid retention capacity and degradation profile, butwith retained biocompatibility. In particular, it is desirable tominimize the degree of modification that is needed to obtain a shapedhyaluronic acid gel product having a desired gel strength, which forinstance can be measured as liquid retention capacity.

Some known soft-tissue augmentation treatments involving implantsoccasionally suffer from the drawback that the implant, or part thereof,migrates away from the desired site of treatment. Another problem withsome known tissue augmentation treatments involving implants is that theimplant is displaced from the desired site of treatment. Implantmigration and displacement are disadvantageous for the patient, sincethey may impair the cosmetic and/or therapeutic outcome of the treatmentand may impede removal of the implant, if this is desired. It is highlybeneficial to maintain the integrity and location of the implant for thedesired time. In order to avoid the aforementioned problems, the gel isrequired to have a certain gel strength in order to resist deformation.This property can be measured using rheometry in the oscillating mode.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method formanufacturing a cross-linked hyaluronic acid product having a desiredshape. A specific object is to provide a method for manufacturing across-linked hyaluronic acid product having a shape that restricts thepossibility for the product to migrate following implantation into asubject.

Another object of the present invention is to provide a method formanufacturing a shaped cross-linked hyaluronic acid product having ahigh biocompatibility, i.e. maintaining the high biocompatibility of thehyaluronic acid when fixed in a desirable and useful shape.

In order to achieve these underlying goals and/or other goals that areevident from the present specification, it has been realized that it isan underlying object of the present invention to provide a method formanufacturing a shaped cross-linked hyaluronic acid product having a lowto moderate degree of modification while at the same time having a highgel strength as shown by low to moderate liquid retention capacity, orswelling degree.

It is a further object of the present invention to provide a method formanufacturing a shaped cross-linked hyaluronic acid product wherein theliquid retention capacity can be controlled or affected by otherparameters than the degree of modification of the hyaluronic acid.

It is an object of the present invention to provide a method formanufacturing a shaped cross-linked hyaluronic acid product wherein ahigh proportion of the bound cross-linking agent(s) is connected in (atleast) two ends, i.e. to achieve a high cross-linking efficiency.

It is a further object of the present invention to provide a shapedcross-linked hyaluronic acid product having a low to moderate degree ofmodification and at the same time a low to moderate liquid retentioncapacity, or swelling degree.

It is an object of the present invention to provide a shapedcross-linked hyaluronic acid product that has a high resistance againstdeformation.

For these and other objects that will be evident from this disclosure,the present invention provides according to a first aspect a method formanufacturing a shaped cross-linked hyaluronic acid product, comprisingthe steps of:

-   -   (i) providing a hyaluronic acid substrate dissolved in a first        liquid medium, which is an aqueous solution, without any        cross-linking;    -   (ii) precipitating the hyaluronic acid substrate by subjecting        it to a second liquid medium comprising an amount of one or more        first water-soluble organic solvent(s) giving precipitating        conditions for hyaluronic acid without any cross-linking;        wherein step (i) and/or step (ii) further comprises arranging        the hyaluronic acid substrate in a desired shape; and    -   (iii) subjecting the non-cross-linked precipitated hyaluronic        acid substrate in the desired shape to a single cross-linking        step in a third liquid medium having a pH of 11.5 or higher and        comprising one or more polyfunctional cross-linking agent(s) and        an amount of one or more second organic solvent(s) giving        precipitating conditions for hyaluronic acid, under suitable        conditions to obtain a precipitated, shaped cross-linked        hyaluronic acid product.

It has been found that this method advantageously allows formanufacturing of shaped cross-linked hyaluronic acid products havinghighly desirable properties. This method provides a good control of thecross-linking since it only occurs in solid (precipitated) phase, andnot in a dissolved phase and/or between method steps. The resultingproduct is unique in that it is a gel with a low swelling degree despitethe low degree of modification of the hyaluronic acid. It is highlysurprising that a gel product having a limited swelling degree at allcan be obtained with this low degree of modification. It is alsosurprising that a process with a single cross-linking reaction canachieve products with such desirable properties. Among manyapplications, this method allows for manufacturing of cross-linkedhyaluronic acid products having a predefined shape that is retainedduring the manufacturing process. The method also allows formanufacturing of biocompatible shaped cross-linked hyaluronic acidproducts.

In a specific embodiment, the first two steps (i) and (ii) occur in theabsence of a cross-linking agent, and the polyfunctional cross-linkingagent is added in the third cross-linking step (iii). This ensures thatthe amount of cross-linking agent is tightly controlled, since nocross-linking agent is reacted or lost in previous steps, e.g. duringthe precipitation step.

In one embodiment, step (i) further comprises arranging the hyaluronicacid substrate solution in a desired shape on a hydrophobic surface; andthe precipitation of the shaped hyaluronic acid substrate in step (ii)occurs on said hydrophobic surface. This is advantageous to avoidclogging of the structures and maintain their shape. The hydrophobicsurface is preferably selected from fluorocarbons, polypropylene (PP),polyethylene terephtalate glycol-modified (PETG), polyethylene (PE), andpolytetrafluoroethylene (PTFE)

In some embodiments, the aqueous solution of step (i) contains 40-100vol % water and 0-60 vol % of lower alkyl alcohol(s). Thereby, anentangled structure can be achieved, which is likely to be advantageousfor obtaining a gel product with desired properties.

In specific embodiments, the second liquid medium of step (ii) contains0-30 vol % water and 70-100 vol % of the first water-soluble organicsolvent(s). In some embodiments, the second liquid medium of step (ii)contains 0-10 vol % water and 90-100 vol % of the first water-solubleorganic solvent(s). A high concentration of the first water-solubleorganic solvent(s) is believed to be advantageous for achieving rapidprecipitation. Thereby, an entangled structure can be achieved, which islikely to be advantageous for obtaining a gel product with desiredproperties.

In certain embodiments, the first water-soluble organic solvent(s) ofstep (ii) is one or more lower alkyl alcohol(s). In some embodiments,the lower alkyl alcohol is ethanol. These organic solvents provide rapidprecipitation.

Step (i) and/or step (ii) further comprises arranging the hyaluronicacid substrate in a desired shape. By the terms “shaped” and “desiredshape” is meant an intentional design which is useful in the finalproduct, i.e. not just a freeze-dried or precipitated hyaluronan powder.In certain embodiments, the shape is selected from the group consistingof a particle, a fibre, a string, a strand, a net, a film, a disc and abead, preferably having an extension of at least 0.5 mm, preferably morethan 1 mm, more preferably more than 5 mm, in at least one dimension. Insome embodiments, the shape of the substrate has an extension of lessthan 5 mm, preferably less than 1 mm, in at least one dimension. Thisfacilitates access for the cross-linking agent(s) to a high number ofthe available binding sites of the precipitated hyaluronic acid productsin the subsequent cross-linking step. In certain embodiments, the shapeof the substrate is longitudinally extended and has a ratio between itslongitudinal extension and its largest lateral extension of 5:1 orhigher, such as 10:1 or higher, e.g. 20:1 or higher, and optionally 100000:1 or lower, such as 25 000:1 or lower, e.g. 100:1 or lower. Sincethe longitudinally extended shape is maintained throughout the methodand in the resulting product, the cross-linked product can be designedto avoid or decrease migration/displacement in vivo, but remains readilyinjectable. In specific embodiments, the shape is a fibre and the ratiobetween its length and its average diameter is 5:1 or higher, such as10:1 or higher, e.g. 20:1 or higher, and optionally 100 000:1 or lower,such as 25 000:1 or lower, e.g. 100:1 or lower.

In certain embodiments, step (ii) involves extruding the hyaluronic acidsubstrate into the second liquid medium comprising an amount of thefirst water-soluble organic solvent(s) giving precipitating conditionsfor hyaluronic acid, thereby allowing the extruded hyaluronic acidsubstrate to form a precipitated fibre in the second liquid medium.

In some embodiments, the third liquid medium of step (iii) contains 0-35vol % water, 65-100 vol % of the second organic solvent(s), and one ormore polyfunctional cross-linking agent(s). In certain embodiments, thesecond organic solvent(s) of step (iii) is one or more lower alkylalcohol(s). In some embodiments, the lower alkyl alcohol is ethanol.

In specific embodiments, the third liquid medium of step (iii) has a pHof 13 or higher. It has surprisingly been realised that performing thecross-linking on a precipitated, shaped substrate at elevated pHprovides shaped gel products with efficient cross-linking, i.e. whereina low degree of modification provides a firm gel with low swellingdegree.

In specific embodiments, the polyfunctional cross-linking agent(s) isindividually selected from the group consisting of divinyl sulfone,multiepoxides and diepoxides. In some embodiments, the polyfunctionalcross-linking agent(s) is individually selected from the groupconsisting of 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanedioldiglycidyl ether (EDDE) and diepoxyoctane. In certain embodiments, thepolyfunctional cross-linking agent is 1,4-butanediol diglycidyl ether(BDDE).

In certain embodiments, the one or more polyfunctional cross-linkingagent(s) provides a single type of cross-links. In specific embodiments,the one or more polyfunctional cross-linking agent(s) provides ethercross-links, which are stable. The present method advantageouslyprovides single cross-linked hyaluronic acid gel products which arestable and can readily be sterilized, e.g. autoclaved.

In some embodiments, the method is further comprising the steps of:

-   -   (iv) subjecting the precipitated cross-linked hyaluronic acid        product to non-precipitating conditions; and    -   (v) isolating the cross-linked hyaluronic acid product in        non-precipitated form.

In certain embodiments, step (v) further comprises sterilizing thecross-linked hyaluronic acid product.

According to another aspect, the present invention provides a shapedcross-linked hyaluronic acid product having a degree of modification of1-40 cross-linking agent units per 1000 disaccharide units, and aswelling degree of 4-300 mL per g hyaluronic acid. This shapedcross-linked hyaluronic acid product has highly useful properties,including a unique combination of a low to moderate degree ofmodification and at the same time a low to moderate swelling degree, orliquid retention capacity. Thereby, it is possible to provide a firm,cross-linked hyaluronic acid product with a desired shape, whilemaintaining the biocompatibility of the native hyaluronic acid. Thecross-linked hyaluronic acid products according to the invention aredesigned in a predefined shape, or structure.

In certain embodiments, the swelling degree is 15-180 mL per ghyaluronic acid.

The modification efficiency (MoE) is a measure of the ratio between theminimum HA concentration (C_(min)), or rigidity/strength, of a gel andits degree of chemical modification by cross-linking agent(s). Inspecific embodiments, the modification efficiency is 10 or higher. Insome embodiments, the modification efficiency is in the range of 20-190or 20-150. Products with a modification efficiency of 10 or higher, suchas in the range of 20-190 or 20-150, combine for the first time a low tomoderate degree of modification and at the same time a low to moderateswelling degree, or liquid retention capacity. Thereby, it is possibleto provide a firm, cross-linked hyaluronic acid product with a desiredshape that is biocompatible and that has a high resistance todeformation.

In certain embodiments, the cross-linker ratio, which describes theproportion of total bound cross-linking agent that has bound two (ormore) disaccharides, is 35% or higher. In specific embodiments, thecross-linker ratio is 40% or higher, and in some even 50% or higher.These shaped products consequently have a low number of cross-linkingagents that do not provide effective cross-links in the product. Thehigh cross-linker ratios allow for a surprisingly low total degree ofmodification in relation to the low to moderate swelling degree, acombination that is advantageous for biocompatibility but sufficient formaintaining the desired shape.

In some embodiments, the shaped hyaluronic acid product is cross-linkedwith one or more polyfunctional cross-linking agent(s) individuallyselected from the group consisting of divinyl sulfone, multiepoxides anddiepoxides. In certain embodiments, the polyfunctional cross-linkingagent(s) is individually selected from the group consisting of1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether(EDDE) and diepoxyoctane. In specific embodiments, the polyfunctionalcross-linking agent is 1,4-butanediol diglycidyl ether (BDDE).

In certain embodiments, the shaped hyaluronic acid product is singlecross-linked. A single cross-linked product has the advantage of beingchemically well defined. It is advantageous that a shaped product with asingle type of cross-links displays such desirable properties. In someembodiments, the shaped hyaluronic acid product is multiplecross-linked. In specific embodiments, the shaped hyaluronic acidproduct is cross-linked with ether cross-links. Shaped ethercross-linked hyaluronic acid gel products according to the invention arestable and can readily be sterilized, e.g. autoclaved.

In certain embodiments, the hyaluronic acid product has a shape selectedfrom the group consisting of a particle, a fibre, a string, a strand, anet, a film, a disc and a bead. In some embodiments, the hyaluronic acidproduct is hollow or has several layers. In certain embodiments, theshape is longitudinally extended and has a ratio between itslongitudinal extension and its largest lateral extension of 5:1 orhigher, such as 10:1 or higher, e.g. 20:1 or higher, and optionally 100000:1 or lower, such as 25 000:1 or lower, e.g. 100:1 or lower.Longitudinally extended cross-linked products can be designed to avoidor decrease migration/displacement in vivo, but remains readilyinjectable. In specific embodiments, the hyaluronic acid product is afibre and the ratio between its length and its width, such as itsaverage diameter, is 5:1 or higher, such as 10:1 or higher, e.g. 20:1 orhigher, and optionally 100 000:1 or lower, such as 25 000:1 or lower,e.g. 100:1 or lower.

In some embodiments, the shaped hyaluronic acid product is present infully swollen state. In the fully swollen state, it is preferred thatthe product has a longitudinally extended shape, and that its largestlateral extension is less than 5 mm, such as less than 1.5 mm, andpreferably less than 0.2 mm. A longitudinally extended cross-linkedproduct with a largest lateral extension of less than 5 mm or even loweris readily injectable. In the fully swollen state, it is furthermorepreferred that the product has a longitudinally extended shape, and thatits longitudinal extension is more than 2 mm, such as more than 25 mm,such as more than 500 mm. A longitudinally extended cross-linked productwith a longitudinal extension of more than 2 mm or higher isadvantageous because it avoids or decreases migration/displacement invivo,

In other embodiments, the shaped hyaluronic acid product is present inpartially swollen or non-swollen state.

In specific embodiments, the shaped hyaluronic acid product isautoclavable. In further specific embodiments, the shaped hyaluronicacid product is autoclaved.

One of the preferred ways of manufacturing a shaped cross-linkedhyaluronic acid product according to the invention is by the methodaccording to the invention.

According to yet another aspect, the present invention provides anaqueous composition comprising a shaped cross-linked hyaluronic acidproduct according to the invention, and optionally a buffering agent.

In certain embodiments, the shaped hyaluronic acid product according tothe invention or the aqueous composition according to the invention isuseful as a medicament or medical device in a medical or surgicalmethod.

According to a further aspect, the present invention provides the use ofa shaped cross-linked hyaluronic acid product according to the inventionor an aqueous composition according to the invention in cosmetic ormedical surgery. Put another way, the present invention provides ashaped cross-linked hyaluronic acid product according to the inventionor an aqueous composition according to the invention for use in cosmeticor medical surgery.

In some embodiments, the use is in cosmetic surgery selected from dermalfilling and body contouring. In some other embodiments, the use is as amedicament in the treatment of, and/or in medical surgery selected from,dermal filling, body contouring, prevention of tissue adhesion,formation of channels, incontinence treatment, and orthopaedicapplications.

According to one aspect, the present invention provides the use of ashaped cross-linked hyaluronic acid product according to the inventionor an aqueous composition according to the invention in drug delivery.In alternative terms, the present invention provides a shapedcross-linked hyaluronic acid product according to the invention or anaqueous composition according to the invention for use in drug delivery.

According to yet another aspect, the present invention provides apre-filled syringe, which is pre-filled with an sterilized, shapedcross-linked hyaluronic acid product according to the invention or ansterilized aqueous composition according to the invention.

According to one aspect, the present invention provides a method oftreatment of a subject undergoing cosmetic or medical surgery, involvingadministration of a shaped cross-linked hyaluronic acid productaccording to the invention or an aqueous composition according to theinvention to a subject in need thereof.

In certain embodiments, the subject is undergoing cosmetic surgeryselected from dermal filling and body contouring. In certain otherembodiments, the subject is undergoing medical surgery, or medicaltreatment, for a condition selected from dermal filling, bodycontouring, prevention of tissue adhesion, formation of channels,incontinence treatment, and orthopaedic applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a fully swelled cross-linked HA net.

FIG. 2 shows 400 MHz ¹H NMR spectra of two enzymatically degraded HA gelformulations.

FIG. 3 shows microscopy images of cross-linked HA gel fibres.

FIG. 4 is a graph showing the change in Swelling Degree (SwD) in g/gduring storage at 60° C. over 14 days.

FIG. 5 shows microscopy images of a cross-linked HA powder (comparativeexample) and a product according to the invention.

FIG. 6 shows microscopy images of cross-linked HA gel fibres.

FIG. 7 shows microscopy images of a cross-linked HA gel passed oncethrough mesh screens (comparative example).

DETAILED DESCRIPTION OF THE INVENTION

According to one aspect, the present invention provides a manufacturingmethod. The method is for manufacturing a shaped cross-linked hyaluronicacid product from a hyaluronic acid substrate.

Unless otherwise provided, the term “hyaluronic acid” encompasses allvariants and combinations of variants of hyaluronic acid, or hyaluronan,of various chain lengths and charge states, as well as with variouschemical modifications That is, the term also encompasses the varioushyaluronate salts of hyaluronic acid, such as sodium hyaluronate.Various modifications of the hyaluronic acid are also encompassed by theterm, such as oxidation, e.g. oxidation of CH₂OH groups to COOH;periodate oxidation of vicinal hydroxyl groups, optionally followed byreduction or imine formation etc; reduction, e.g. reduction of COOH toCH₂OH; sulphation; deamidation, optionally followed by deamination oramide formation with new acids; esterification; substitutions withvarious compounds, e.g. using a cross-linking agent or a carbodiimide;including coupling of different molecules, such as proteins, peptidesand active drug components, to hyaluronic acid; and deacetylation. It iswell known to the skilled person that the various forms of hyaluronicacid have different chemical properties that have to be taken intoaccount during chemical modification and analysis. For instance, if itis desired to obtain a solution of hyaluronic acid having a certain pH,the acidity of the material to be dissolved, the acidity of thedissolving liquid and any buffering capacity will all affect theresulting pH of the solution.

It is preferred that the hyaluronic acid substrate is a hyaluronic acidor hyaluronate salt without chemical modifications, i.e. which has notbeen subjected to cross-linking or other modifications prior to thepresent manufacturing method.

The hyaluronic acid can be obtained from various sources of animal andnon-animal origin. Sources of non-animal origin include yeast andpreferably bacteria. The molecular weight of a single hyaluronic acidmolecule is typically in the range of 1.5-3 MDa, but other ranges ofmolecular weights are possible, e.g. 0.5-10 MDa.

The product that is manufactured by the method is a shaped cross-linkedhyaluronic acid. The method provides cross-links between the hyaluronicacid chains when they have been arranged in a desirable shape, whichcreates a continuous shaped network of hyaluronic acid molecules whichis held together by the covalent cross-links, physical entangling of thehyaluronic acid chains and various interactions, such as hydrogenbonding, van der Waals forces and electrostatic interactions. The shapedcross-linked hyaluronic acid product according to the invention is agel, or a hydrogel. That is, it can be regarded as a water-insoluble,but substantially dilute, cross-linked system of hyaluronic acidmolecules when subjected to a liquid, typically an aqueous liquid.

The resulting shaped cross-linked hyaluronic acid product is preferablybiocompatible. This implies that no, or only very mild, immune responseoccurs in the treated individual. In the Examples, there is provided amethod of determining the biocompatibility of a hyaluronic acid product,and results from testing the biocompatibility of a cross-linkedhyaluronic acid product according to the invention in rats.

The method according to the invention is comprising at least threesteps: a preparation step, a precipitation step, and a cross-linkingstep. In certain embodiments, the method is consisting of these threesteps.

In the first method step, a hyaluronic acid substrate is provided. Asset out above, the term “hyaluronic acid substrate” encompasses allvariants and combinations of variants of hyaluronic acid, or hyaluronan,of various chain lengths and charge states, as well as with variouschemical modifications. It is preferable that the hyaluronic acidsubstrate is a chemically unmodified hyaluronic acid or hyaluronatesalt, preferably sodium hyaluronate, having an average molecular weightin the range of 0.5-10 MDa, preferably 0.8-5 MDa, more preferably 1.5-3MDa or 2-3 MDa. It is preferred that the hyaluronic acid is obtainedfrom non-animal origin, preferably bacteria.

The hyaluronic acid substrate is dissolved in a first liquid medium,which is an aqueous solution. By the terms “dissolved” and “solution” isunderstood that the hyaluronic acid substrate is present in ahomogeneous mixture with a liquid, in which mixture energeticallyfavourable interactions occur. Addition of liquid to the solution lowersthe concentration of the dissolved hyaluronic acid substrate. Thesolution is aqueous, i.e. it contains water. The aqueous solution maysimply consist of the hyaluronic acid substrate dissolved in water. Itis preferable that the aqueous solution contains 40-100 vol % water and0-60 vol % of lower alkyl alcohol(s). The term “lower alkyl alcohol”includes primary, secondary and tertiary alkyl alcohols having from oneto six carbon atoms, i.e. C₁₋₆ alkyl alcohols. Specific examples oflower alkyl alcohols include methanol, ethanol, denatured spirit,n-propanol, isopropanol, n-butanol, isobutanol, and t-butanol. Preferredlower alkyl alcohols are methanol and ethanol, in particular ethanol,due to price, availability and easy handling. The lower alkyl alcoholconcentration is preferably in the range of 0-40%, such as 0-20%, 10-30%or 20-40% ethanol, with corresponding adjustments to the watercomponent. The pH of this aqueous solution is suitably 6 or higher, suchas 9 or higher.

Optionally, the first method step further involves arranging thehyaluronic acid substrate in a desired shape, such as a particle, afibre, a string, a strand, a net, a film, a disc and a bead, whichoptionally is hollow or contain different layers of material. This maybe accomplished in various ways, e.g. moulding and extrusion. Extrusionof the hyaluronic acid substrate typically involves pressing thehyaluronic acid substrate solution through an opening of desired size.The extruded hyaluronic acid substrate spontaneously forms aprecipitated fibre, string or strand. The dimensions, e.g. thethickness, of the fibre, string or strand can be controlled by varyingthe dimension or type of opening, e.g. using various opening diametersin the range of 0.1-2 mm or 14-30 G, the extrusion pressure, theextrusion speed and/or the hyaluronic acid concentration. By using othertypes of orifices and chinks, different shapes or structures can beproduced. For instance, the hyaluronic acid can be precipitated as afilm, a net, discs or beads.

In a preferred embodiment, the hyaluronic acid substrate solution isarranged in the desired shape on a hydrophobic surface, and thesubsequent precipitation of the shaped hyaluronic acid substrate occurson said hydrophobic surface. This is advantageous to avoid clogging ofthe shapes structures and to maintain the desired shape until it isfixed by the subsequent cross-linking step. Suitable hydrophobicsurfaces are well known to the skilled person and include e.g.fluorocarbons, polypropylene (PP), polyethylene terephtalateglycol-modified (PETG), polyethylene (PE), and polytetrafluoroethylene(PTFE).

This shape can be maintained throughout the manufacturing method and inthe final product. It is preferred that the shape has an extension ofless than 5 mm, preferably less than 1 mm, such as less than 0.5 mm oreven less than 0.2 mm, in at least one dimension when the hyaluronicacid substrate is in precipitated form. This facilitates access for thecross-linking agent(s) to a high number of available binding sites ofthe precipitated hyaluronic acid products in the subsequentcross-linking step. It is also preferred that the shape islongitudinally extended and has a ratio between its longitudinalextension and its largest lateral extension of 5:1 or higher, such as10:1 or higher, e.g. 20:1 or higher, and optionally 100 000:1 or lower,such as 25 000:1 or lower, e.g. 100:1 or lower. Since the longitudinallyextended shape is maintained throughout the method and in the resultingproduct, the cross-linked product can be designed to avoid or decreasemigration/displacement in vivo, but remains readily injectable. Asuitable example of such shape is a fibre and the ratio between itslength and its width is 5:1 or higher, such as 10:1 or higher, e.g. 20:1or higher, and optionally 100 000:1 or lower, such as 25 000:1 or lower,e.g. 100:1 or lower. A preferred composition is comprising cross-linkedstrand/fibre-shaped hyaluronic acid products according to the invention,wherein more than 50% of the products have a ratio between itslongitudinal extension and its largest lateral extension of 5:1 orhigher, such as 10:1 or higher, e.g. 20:1 or higher, and optionally 100000:1 or lower, such as 25 000:1 or lower, e.g. 100:1 or lower.

By way of example, a cross-linked hyaluronic acid product according tothe invention with a single strand or fibre shape filling up a 20 mLsyringe may have a thickness of 1 mm and a length of 25 m in swelledstate, i.e. a ratio between its longitudinal extension and its largestlateral extension of 25000:1. An example of a preferred composition iscomprising cross-linked strand/fibre-shaped hyaluronic acid productsaccording to the invention, wherein more than 50% of the products have alongitudinal extension of more than 2 mm and a largest lateral extensionof less than 0.2 mm, i.e. a ratio of 10:1 or higher.

The first method step is carried out without cross-linking, and this maybe achieved by omitting cross-linking agents in this step and/orproviding conditions that are not suitable for cross-linking. It isimportant to ensure that cross-linking does not occur until thepreferred shape has been attained. This is advantageous for obtainingand maintaining a desired shape of the final product, since the shapingof the substrate is not limited by pre-existing cross-links, and allcross-links produced in the third step are directed to maintaining thedesired shape of the product. It is preferred that the first step occursin the absence of a cross-linking agent. This provides a good control ofthat the cross-linking does not occur in a dissolved phase and/orbetween method steps. It also ensures that the amount of cross-linkingagent is tightly controlled and that the resulting products arehomogenous in quality, since no cross-linking agent is reacted or lostin previous steps. Avoiding cross-linking, and in particular theaddition of cross-linking agent in this step is useful to obtain amanufacturing process that is suitable for scaling up to an industrialscale and for providing products with homogenous quality.

In the second method step, the hyaluronic acid substrate is precipitateddue to reduction of the solubility of the hyaluronic acid substrate.This is achieved by subjecting the hyaluronic acid substrate to a secondliquid medium in which it is insoluble. The second liquid mediumcomprises an amount of one or more first water-soluble organicsolvent(s) giving precipitating conditions for hyaluronic acid. Theresulting solid precipitate falls out of the solute phase and cantypically be separated from the remaining liquid by filtration,decanting, centrifugation, or manually using a pair of tweezers or thelike. In one preferred embodiment, the precipitated hyaluronic acidsubstrate is also removed from the medium and dried. The precipitate canalso be maintained suspended in the second liquid medium. Thus, inanother preferred embodiment, the precipitated hyaluronic acid substrateis not subjected to drying. It is advantageous to achieve theprecipitation of the hyaluronic acid substrate in a rapid fashion, e.g.by extruding or immersing the hyaluronic acid substrate in the secondliquid medium in which it is insoluble.

The organic solvents that are used according to the invention arecarbon-containing solvents and may exhibit a varying degree of polarity.Although termed “solvents”, it shall be understood that these organicsolvents are utilized for balancing and shifting the solubility ofhyaluronic acid during the manufacturing method. The hyaluronic acid mayvery well be dissolved in an organic solvent at a certain organicsolvent concentration interval, but falls out and forms a precipitatewhen the organic solvent concentration is increased. For instance, thehyaluronic acid can be dissolved in a 50/50 (vol/vol) mixture of anorganic solvent, e.g. a lower alkyl alcohol, and water, but falls outand forms a precipitate in a 90/10 (vol/vol) mixture. When subjected tonon-precipitating conditions, e.g. a 50/50 or a 0/100 mixture, thehyaluronic acid returns to the non-precipitated, dissolved state. Theskilled person is well aware that other factors may have an impact onthe limiting organic solvent(s) concentration for precipitation ofhyaluronic acid, such as temperature, pH, ion strength and type oforganic solvent(s). The limiting concentration for precipitation ofhyaluronic acid under given conditions is well known or can easily bedetermined by a skilled person in the field. By way of example, thelimiting concentration for precipitation of hyaluronic acid (in mixtureof water and ethanol) is approximately 70% ethanol.

Without being limited thereto, the organic solvents according to theinvention can be selected from the group consisting of pentane, hexane,cyclohexane, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide,ethyl acetate, acetamide, diethyl ether, tetrahydrofurane, acetonitrile,methyl ethyl ketone, acetone, lower alkyl alcohols, e.g. methanol,ethanol, propanol, isopropanol and butanol, It is preferable that theorganic solvents according to the invention are water-soluble. Apreferred group of organic solvents is the lower alkyl alcohols. Theterm lower alkyl alcohol includes primary, secondary and tertiary alkylalcohols having from one to six carbon atoms, i.e. C₁₋₆ alkyl alcohols.Specific examples of lower alkyl alcohols include methanol, ethanol,denatured spirit, n-propanol, isopropanol, n-butanol, isobutanol, andt-butanol. Preferred lower alkyl alcohols are methanol and ethanol, inparticular ethanol, due to price, availability and easy handling.

It is suitable that the second liquid medium of the second method stepis an aqueous medium, i.e. that it contains water to some extent. It ispreferred that the second liquid medium contains 0-30 vol % water and70-100 vol % of the first water-soluble organic solvent(s), preferably0-10 vol % water and 90-100 vol % of the first water-soluble organicsolvent(s). In certain embodiments, the concentration of the firstwater-soluble organic solvent(s) may be as high as 95%, such as 99% oreven 99.5%, e.g. 99% methanol or ethanol. A high concentration of thefirst water-soluble organic solvent(s) is believed to be advantageousfor achieving rapid precipitation. Thereby, an entangled structure canbe achieved, which is likely to be advantageous for obtaining a gelproduct with desired properties.

The second method step is carried out without cross-linking, and thismay be achieved by omitting cross-linking agents in this step and/orproviding conditions that are not suitable for cross-linking. It isimportant to ensure that cross-linking does not occur until thepreferred shape has been attained. This is advantageous for obtainingand maintaining a desired shape of the final product, since the shapingof the substrate is not limited by pre-existing cross-links, and allcross-links produced in the third step are directed to maintaining thedesired shape of the product. It is preferred that the second stepoccurs in the absence of a cross-linking agent. This provides a goodcontrol of that the cross-linking does not occur in a dissolved phaseand/or between method steps. It also ensures that the amount ofcross-linking agent is tightly controlled and that the resultingproducts are homogenous in quality, since no cross-linking agent isreacted or lost in previous steps. Avoiding cross-linking, and inparticular the addition of cross-linking agent, in this step is usefulto obtain a manufacturing process that is suitable for scaling up to anindustrial scale and for providing products with homogenous quality.

Optionally, the second method step further involves arranging thehyaluronic acid substrate in a desired shape, such as a particle, afibre, a string, a strand, a net, a film, a disc and a bead, whichoptionally is hollow or contain different layers of material. This maybe accomplished in various ways, e.g. moulding and extrusion. This shapecan be maintained throughout the manufacturing method and in the finalproduct. It is preferred that the shape of the precipitated substratehas an extension of less than 5 mm, preferably less than 1 mm, such asless than 0.5 mm or even less than 0.2 mm, in at least one dimensionwhen the hyaluronic acid substrate is in precipitated form. Thisfacilitates access for the cross-linking agent(s) to a high number ofavailable binding sites of the precipitated hyaluronic acid products inthe subsequent cross-linking step. It is also preferred that the shapeof the precipitated substrate is longitudinally extended and has a ratiobetween its longitudinal extension and its largest lateral extension of5:1 or higher, such as 10:1 or higher, e.g. 20:1 or higher, andoptionally 100 000:1 or lower, such as 25 000:1 or lower, e.g. 100:1 orlower. Since the longitudinally extended shape is maintained throughoutthe method and in the resulting product, the cross-linked product can bedesigned to avoid or decrease migration/displacement in vivo, butremains readily injectable. A suitable example of such shape is a fibreand the ratio between its length and its width is 5:1 or higher, such as10:1 or higher, e.g. 20:1 or higher, and optionally 100 000:1 or lower,such as 25 000:1 or lower, e.g. 100:1 or lower. By way of example, across-linked hyaluronic acid product according to the invention with asingle strand or fibre shape filling up a 20 mL syringe may have athickness of 1 mm and a length of 25 m in swelled state, i.e. a ratiobetween its longitudinal extension and its largest lateral extension of25000:1.

The second method step may involve extrusion of the hyaluronic acidsubstrate into the second liquid medium, which is comprising an amountof the first water-soluble organic solvent(s) giving precipitatingconditions for hyaluronic acid. This is typically involving pressing thehyaluronic acid substrate solution through an opening of desired sizeinto the second liquid medium. The extruded hyaluronic acid substratespontaneously forms a precipitated fibre, string or strand in the secondliquid medium. The dimensions, e.g. the thickness, of the fibre, stringor strand can be controlled by varying the dimension or type of opening,e.g. using various opening diameters in the range of 0.1-2 mm or 14-30G, the extrusion pressure, the extrusion speed and/or the hyaluronicacid concentration. By using other types of orifices and chinks,different shapes or structures can be produced. For instance, thehyaluronic acid can be precipitated as a film, a net, discs or beads.When a fibre, string or strand is formed, it is preferred that the ratiobetween its length and its average diameter is 5:1 or higher, such as10:1 or higher, e.g. 20:1 or higher, and optionally 100 000:1 or lower,such as 25 000:1 or lower, e.g. 100:1 or lower. An advantage with thefibre/string/strand shape is that the fibres/strings/strands themselvescan be entangled at the macroscopic level, causing a coil or ball effectwhich may be advantageous, e.g. for maintaining the integrity of animplant.

In the third method step, the precipitated hyaluronic acid substrate isfor the first time subjected to cross-linking in a third liquid medium.The term “cross-linking” refers to introduction of stable covalent links(cross-links) between (at least) two different hyaluronic acid chains or(at least) two distinct sites of a single hyaluronic acid chain, whichcreates a continuous network of hyaluronic acid molecules. Thecross-link may simply be a covalent bond between two atoms in thehyaluronic acid chains, e.g. an ether bond between two hydroxyl groups,or an ester bond between a hydroxyl group and a carboxyl group. Thecross-link may also be a linker molecule that is covalently bound to twoor more atoms of different hyaluronic acid chains or distinct sites of asingle hyaluronic acid chain. Although cross-linking can occurspontaneously under certain conditions, the cross-linking typicallyinvolves use of a cross-linking agent, one or more, which facilitatesand speeds up the process. When the cross-linking is accomplished, thecross-linking agent(s) may be entirely or partially linked to thehyaluronic acid or it may be degraded. Any remaining residuals ofnon-bound cross-linking agent can be removed after the cross-linking.

It is important to ensure that cross-linking does not occur until thepreferred shape has been attained. This is advantageous for obtainingand maintaining a desired shape of the final product, since the shapingof the substrate is not limited by pre-existing cross-links, and allcross-links produced in the third step are directed to maintaining thedesired shape of the product. It is preferred that the first two stepsoccur in the absence of a cross-linking agent, and that a cross-linkingagent is added in the third cross-linking step. This provides a goodcontrol of the cross-linking since it only occurs in solid(precipitated) phase, and not in a dissolved phase and/or between methodsteps. It also ensures that the amount of cross-linking agent is tightlycontrolled and that the resulting products are homogenous in quality,since no cross-linking is reacted or lost in previous steps, e.g. duringthe precipitation step. Altogether, focusing the cross-linking, and inparticular the addition of cross-linking agent, to the final step isuseful to obtain a manufacturing process that is suitable for scaling upto an industrial scale and for providing products with homogenousquality.

The third liquid medium contains one or more cross-linking agent(s) thatis polyfunctional, i.e. it has two or more reaction sites for formingcovalent bonds to the hyaluronic acid molecules that are beingcross-linked. It is preferred that the cross-linking agent(s) that isused in this third step is bifunctional, i.e. it has two reaction sitesfor forming covalent bonds to the hyaluronic acid molecules that arebeing cross-linked. Without being limited thereto, useful polyfunctionalcross-linking agents include divinyl sulfone, multiepoxides anddiepoxides, such as 1,4-butanediol diglycidyl ether (BDDE),1,2-ethanediol diglycidyl ether (EDDE) and diepoxyoctane, preferablyBDDE. It is desirable that the one or more polyfunctional cross-linkingagent(s) provide ether cross-links. The present method advantageouslyprovides ether cross-linked hyaluronic acid gel products which arestable and can readily be sterilized, e.g. autoclaved. Ester cross-linksare less stable and will more easily be hydrolysed.

The cross-linking of the third method step is performed underprecipitating conditions so that the shaped hyaluronic acid substrate isprecipitated. In particular, the available surface of the hyaluronicacid molecules is in precipitated form due to the precipitatingconditions. The third liquid medium contains an amount of one or moresecond organic solvent(s) giving precipitating conditions for hyaluronicacid, which amount and/or organic solvent(s) may be the same as ordifferent to what was used in the second liquid medium of the secondmethod step to precipitate the shaped hyaluronic acid substrate.

As detailed above, it shall be understood that the organic solvents areutilized for balancing and shifting the solubility of hyaluronic acidduring the manufacturing method. The skilled person is well aware thatother factors may have an impact on the limiting organic solvent(s)concentration for precipitation of hyaluronic acid, such as temperature,pH, ion strength and type of organic solvent(s). The limitingconcentration for precipitation of hyaluronic acid under givenconditions is well known or can easily be determined by a skilled personin the field.

Using this method, it is also possible to obtain shaped cross-linkedhyaluronic acid products with a single cross-linking reaction in thethird method step. Depending on the choice and number of cross-linkingagents in the single reaction step, the resulting shaped product may besingle cross-linked, i.e. containing essentially a single type ofcross-links, preferably stable ether cross-links, or multiplecross-linked, i.e. containing at least two different types ofcross-links, preferably including stable ether cross-links. It issurprising that a process with a single cross-linking reaction canachieve shaped products with such desirable properties.

Without being limited thereto, the organic solvents according to theinvention can be selected from the group consisting of pentane, hexane,cyclohexane, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide,ethyl acetate, acetamide, diethyl ether, tetrahydrofurane, acetonitrile,methyl ethyl ketone, acetone, lower alkyl alcohols, e.g. methanol,ethanol, propanol, isopropanol and butanol, It is preferable that theorganic solvents according to the invention are water-soluble. Apreferred group of organic solvents is the lower alkyl alcohols. Theterm lower alkyl alcohol includes primary, secondary and tertiary alkylalcohols having from one to six carbon atoms, i.e. C₁₋₆ alkyl alcohols.Specific examples of lower alkyl alcohols include methanol, ethanol,denatured spirit, n-propanol, isopropanol, n-butanol, isobutanol, andt-butanol. Preferred lower alkyl alcohols are methanol and ethanol, inparticular ethanol, due to price, availability and easy handling.

It is suitable that the third liquid medium of the third method step isan aqueous medium, i.e. that it contains water to some extent. It ispreferred that the third liquid medium, in addition to the cross-linkingagent(s), contains 0-35 vol % water and 65-100 vol % of the secondwater-soluble organic solvent(s), preferably 20-35 vol % water and 65-80vol % of the second water-soluble organic solvent(s).

The third liquid medium has a pH of 11.5 or higher, i.e. thecross-linking is performed at a pH of 11.5 or higher As the skilledperson is well aware, the acidity of the hyaluronic acid startingmaterial has to be taken into account to obtain a desired pH in thethird liquid medium. This may be accomplished by addition of acids,bases or buffer systems with suitable buffering capacity. A preferred pHregulator is a strong base, such as sodium hydroxide. It has been foundthat a basic pH is advantageous when cross-linking the precipitatedhyaluronic acid molecules. It is preferred that the third liquid mediumof the third step has a pH of 13 or higher. In a liquid mediumcontaining both water and organic solvent(s), the measured pH may differfrom the theoretical pH due to the type of solvent(s) and amount ofrespective solvent(s). Therefore, the term apparent pH (pH_(app)) isintroduced to indicate the pH that is measured using standard pHmeasurement equipment under the given conditions. An accurate pH valuecan readily be determined by e.g. titration.

The cross-linking in the third method step produces a shapedcross-linked hyaluronic acid product according to the invention. Theshaped cross-linked hyaluronic acid product according to the inventionis a gel, or a hydrogel. That is, it can be regarded as awater-insoluble, but substantially dilute cross-linked system ofhyaluronic acid molecules when subjected to a liquid, typically anaqueous liquid. Since the cross-linking of the third method step isperformed under precipitating conditions, both the shaped hyaluronicacid substrate and the shaped cross-linked product are precipitated. Inparticular, the available surface of both the shaped hyaluronic acidsubstrate and the shaped cross-linked product is in precipitated formdue to the precipitating conditions of this third method step. Thecross-linking reaction is allowed to proceed under suitable conditionsuntil a desired amount of cross-linking agent(s) has reacted with theshaped hyaluronic acid substrate. The amount of cross-linking agent(s)that has bound to the hyaluronic acid can be quantified and reported asthe degree of modification (MoD), i.e. the molar amount of boundcross-linking agent(s) relative to the total number of repeating HAdisaccharide units. It is preferred that the cross-linking reaction isallowed to proceed until the degree of modification (MoD) of thecross-linked hyaluronic acid product is in the range of 1-40cross-linking agent units per 1000 disaccharide units (0.1-4%),preferably 1-10 cross-linking agent units per 1000 disaccharide units(0.1-1%). The required reaction time is governed by several factors,such as hyaluronic acid concentration, cross-linking agent(s)concentration, temperature, pH, ion strength and type of organicsolvent(s). These factors are all well known to the person skilled inthe art, who easily can adjust these and other relevant factors andthereby provide suitable conditions to obtain a degree of modificationin the range of 0.1-4% and verify the resulting product characteristicswith respect to the degree of modification. The cross-linking of thethird step occurs for at least 2 h, preferably at room temperature forat least 24 h.

Any residual non-bound cross-linking agent(s) can be removed when theshaped precipitated product is separated from the cross-linking medium.The shaped cross-linked hyaluronic acid product can be further purifiedby additional washing steps with a suitable washing liquid, e.g. water,methanol, ethanol, saline or mixtures and/or combinations thereof.

The manufacturing method according to the invention thus allows for aproduction of predefined physical forms, or structures, of thecross-linked hyaluronic acid products, such as a particle, a fibre, astring, a strand, a net, a film, a disc or a bead. Structures that arelongitudinally extended, or rod-shaped, and have a ratio between theirlongitudinal extension and their largest lateral extension of 5:1 orhigher, such as 10:1 or higher, e.g. 20:1 or higher, and optionally 100000:1 or lower, such as 25 000:1 or lower, e.g. 100:1 or lower, areparticularly useful as medical or cosmetic implants, because they can bedimensioned to avoid migration by having a sufficient length, and can atthe same time readily be administered by injection through a needle dueto the limited width. By way of example, a cross-linked hyaluronic acidproduct according to the invention with a single strand or fibre shapefilling up a 20 mL syringe may have a thickness of 1 mm and a length of25 m in swelled state, i.e. a ratio between its longitudinal extensionand its largest lateral extension of 25000:1.

The predefined structures can optionally be hollow or consist ofmultiple layers. The space created in a hollow predefined structure isoptionally filled with HA, which may be modified, such as cross-linkedor substituted with other compounds. One or more of the multiple layersin a predefined layer structure may consist of cross-linked ornon-cross-linked HA, which may be chemically modified by substitutionwith other compounds. This may be accomplished by arranging thehyaluronic acid substrate in a desired shape in the first method step,e.g. by extrusion or moulding, or in the second method step, e.g. byextrusion of the dissolved hyaluronic acid substrate into aprecipitating medium, e.g. ethanol. The acquired shape can be maintainedthroughout the manufacturing method and in the final product.

Optionally, the manufacturing method further involves a fourth step ofsubjecting the shaped precipitated cross-linked hyaluronic acid productto non-precipitating conditions. That is, the method is in certainembodiments comprising four steps, or alternatively consisting of foursteps. This typically involves subjecting the shaped cross-linkedhyaluronic acid product to a liquid medium and allowing it to return tonon-precipitated state. The liquid medium is typically water, saline ormixtures and/or combinations thereof, optionally with non-precipitatingconcentrations of an organic solvent, e.g. methanol or ethanol. Due tothe cross-linking, the resulting shaped hyaluronic acid product is acontinuous network of interconnected and entangled hyaluronic acidchains which under non-precipitating conditions absorbs liquid (swells)and forms a gel. The swelling can be allowed to proceed until the gel isfully swollen and no further liquid can be absorbed, or it can beinterrupted at an earlier stage to obtain a partially swollen gel. Apartially swollen gel can be useful as an intermediate for furtherprocessing of the gel, for instance further mechanical production of gelstructures of a desired size and shape can be performed. By way ofexample, a film can be cut into particles, slices or pieces, gel fibrescan be cut into shorter fragments, well defined irregular shapes can bedesigned from a film, etc. The cross-linked HA fibres, strings orstrands can also be woven together to form a net or a film aftercompleted cross-linking, before or after drying. It may also beconvenient to use a partially swollen shaped gel product duringimplantation thereof at a desired site to facilitate administration andminimize patient discomfort and to enhance the lifting capacity by useof the remaining swelling capacity.

When the shaped gel product is subjected to non-precipitating conditionsin an excess of liquid, it is also possible to determine its maximumswelling degree, or inversely its minimum hyaluronic acid concentration(C_(min)), i.e. the hyaluronic acid concentration when the gel productis fully swollen. Using the manufacturing method according to theinvention, it is possible to obtain a swelling degree of 4-300 mL per ghyaluronic acid, and preferably 15-180 mL per g hyaluronic acid. Thisimplies C_(min) values in the range of 0.3-25% (w/v), preferably 0.6-7%(w/v), corresponding to 3-250 mg/g, preferably 6-70 mg/g. It is highlyadvantageous that the desired swelling degree (or C_(min) value) can beachieved with a minimal degree of modification, but the traditional wayof regulating the swelling degree is by means of varying the degree ofmodification. The present manufacturing method therefore provides a newconcept for regulating the swelling capacity of a shaped gel product,which surprisingly enables production of firm shaped gels with auniquely high C_(min) value (low swelling degree) in relation to the lowdegree of modification of the gel.

The modification efficiency (MoE) is a measure of the ratio between theminimum HA concentration (C_(min)), or rigidity/strength, of a gel andits degree of chemical modification by cross-linking agent(s). Using themanufacturing method according to the present invention, it is possibleto obtain a cross-linked hyaluronic acid product having a modificationefficiency of 10 or higher, preferably in the range of 10-200, such as20-150 or 20-190. Without desiring to be limited to any specific theory,it is contemplated that the beneficial properties of the gel are theresult of a surprisingly high degree of effective cross-linking, i.e. ahigh degree of the bound cross-linking agent(s) (cross-linker ratio,typically 0.35 or 35% or higher, such as 40% or higher or even 50% orhigher) is in fact bound to the hyaluronic acid at two (or more) sites,in combination with effective positioning of the cross-links for thedesired purpose, and probably an extremely high degree of retainedentanglement. In contrast to what a skilled person would expect from thelow degree of modification of the resulting hyaluronic acid product, themethod according to the invention surprisingly provides a gel with highrigidity/strength. Under any circumstances, the method according to theinvention provides a useful way of further regulating the swellingdegree in relation to the degree of modification. The method is alsovery suitable for continuous operation, which is advantageous forlarge-scale production.

Optionally, the manufacturing method also involves a final step ofisolating the cross-linked hyaluronic acid product. That is, the methodis in certain embodiments comprising four or five steps, oralternatively consisting of four or five steps. Depending on whether theproduct is held under precipitating conditions or has been subjected tonon-precipitating conditions, this step may involve isolating theproduct in precipitated form or in non-precipitated precipitated form.The isolated, precipitated or non-precipitated, product can then besubjected to sterilization so as to obtain a sterile cross-linkedhyaluronic acid product.

If desired, other substances, such as local anaesthetics (e.g. lidocainehydrochloride) anti-inflammatory drugs, antibiotics and other suitablesupportive medications, e.g. bone growth factors or cells, may be addedafter the cross-linked hyaluronic acid product has been obtained.

According to one aspect, the invention provides a shaped cross-linkedhyaluronic acid product. According to one embodiment, the product ismanufactured, or can be manufactured, by the manufacturing method of theinvention. The shaped cross-linked hyaluronic acid product according tothe invention is a gel, or a hydrogel. That is, it can be regarded as awater-insoluble, but substantially dilute cross-linked system ofhyaluronic acid molecules when subjected to a liquid, typically anaqueous liquid. The gel is mostly liquid by weight and can e.g. contain90-99.9% water, but it behaves like a solid due to a three-dimensionalcross-linked hyaluronic acid network within the liquid. Due to itssignificant liquid content, the shaped gel is structurally flexible andsimilar to natural tissue, which makes it very useful as a scaffold intissue engineering and for tissue augmentation. It is the cross-linksand their attachment positions at the hyaluronic acid molecules that,together with the natural entanglement of the hyaluronic acid chains,give the gel its structure and properties, which are intimately relatedto its swelling degree.

The amount of attached cross-linking agent(s) can be quantified by andreported as the degree of modification (MoD), i.e. the molar amount ofbound cross-linking agent(s) relative to the total number of repeatingHA disaccharide units. It is preferred that the cross-linked hyaluronicacid product according to the invention has a degree of modification of1-40 cross-linking agent units per 1000 disaccharide units (0.1-4%),preferably 1-10 cross-linking agent units per 1000 disaccharide units(0.1-1%). The effectiveness of the cross-linking reaction is shown bythe amount of attached cross-linking agent(s) that is connected in (atleast) two ends to one (or more) hyaluronic acid chains and is reportedas the cross-linker ratio (CrR). It is preferable that the productaccording to the invention has a cross-linker ratio of 35% or higher,preferably 40% or higher, more preferably 50% or higher, such as in theranges of 35-80%, 40-80% and 50-80%. These products consequently have alow number of cross-linking agents that do not provide effectivecross-links in the product. The high cross-linker ratios allow for asurprisingly low total degree of modification in relation to the gelstrength, which in turn is advantageous to ensure high biocompatibility.

Another characteristic of a gel is its capacity to absorb water until itis fully swollen. Further addition of liquid will not dilute the gelfurther, i.e. the gel cannot be indefinitely diluted like a solution offree molecules. When the gel is subjected to non-precipitatingconditions, it is also possible to determine its swelling degree, orinversely its minimum concentration (C_(min)), i.e. the hyaluronic acidconcentration when the gel product is fully swollen. Harder(low-swelling) gels are generally less viscous, more elastic andexpected to have a longer half-life in vivo than softer (high-swelling)gels. However, harder gels may be recognised as foreign materials by thebody if they are highly chemically modified. It is preferred that theproduct according to the invention has a swelling degree of 4-300 mL perg hyaluronic acid, and preferably 15-180 mL per g hyaluronic acid. Thisimplies C_(min) values in the range of 0.3-25% (w/w), i.e. 3-250 mg/g,such as 0.6-7% (w/w), i.e. 6-70 mg/g. It is preferred that the C_(min)value is 0.6-5% (w/v), i.e. 6-50 mg/mL.

It is preferred that the shaped cross-linked hyaluronic acid productaccording to the inventions is cross-linked with one or morecross-linking agent(s) that is polyfunctional, i.e. it has two or morereaction sites for forming covalent bonds to the hyaluronic acidmolecules that are being cross-linked. It is preferred that thecross-linking agent(s) is bifunctional, i.e. it has two reaction sitesfor forming covalent bonds to the hyaluronic acid molecules that arebeing cross-linked. Without being limited thereto, useful polyfunctionalcross-linking agents include divinyl sulfone, multiepoxides anddiepoxides, such as 1,4-butanediol diglycidyl ether (BDDE),1,2-ethanediol diglycidyl ether (EDDE) and diepoxyoctane, preferablyBDDE. It is desirable that the hyaluronic acid gel product iscross-linked with ether cross-links, which are stable.

The shaped hyaluronic acid gel product can be multiple cross-linked,i.e. containing at least two different types of bonds, preferablyincluding ether bonds. It is preferred that the hyaluronic acid productis single cross-linked, i.e. containing essentially a single type ofcross-links, preferably ether cross-links. A single cross-linked producthas the advantage of being chemically well defined. It is advantageousthat a product with a single type of cross-links displays such desirableproperties. In specific embodiments, the hyaluronic acid product iscross-linked with ether cross-links, which provides a stable andautoclavable product.

It is highly advantageous that the desired swelling degree (or C_(min)value) of the shaped product is obtained with a minimal degree ofmodification, although the traditional way of regulating the swellingdegree is by means of varying the degree of modification. The mainreason for minimizing the degree of modification is to ensure that thebiocompatibility of the gel is high, but the skilled person is wellaware of other advantages. The shaped product is characterised by auniquely high C_(min) value (low swelling degree) in relation to thedegree of modification of the gel. The modification efficiency (MoE) isa measure of the ratio between the minimum HA concentration (C_(min)),that reflects the rigidity and the strength of a gel, and the degree ofchemical modification of the gel with cross-linking agent(s). The shapedcross-linked hyaluronic acid product according to the invention has amodification efficiency of 10 or higher, preferably in the range of10-200, such as in the range of 20-150 or 20-190. Products with amodification efficiency of 10 or higher, such as in the range of 20-190,combine for the first time a low to moderate degree of modification andat the same time a low to moderate swelling degree, or liquid retentioncapacity. Thereby, it is possible to provide a firm, cross-linkedhyaluronic acid product that is biocompatible and that has a highresistance to deformation.

Furthermore, it is preferable that the shaped cross-linked hyaluronicacid gel products according to the invention are viscoelastic. Thisimplies that the gel products exhibit a combination of viscous andelastic properties. As is well known by the skilled person, theviscoelastic properties can be determined with a rheometer. Inoscillating mode, the elastic modulus (G′) and the viscous modulus (G″)can be determined at a frequency of 0.1 or 1 Hz. For certainviscoelastic gel products according to the invention, it is preferredthat the following relationship is satisfied:

${0.1 \leq \frac{G^{\prime}}{\left( {G^{''} + G^{\prime}} \right)} \leq 0.98},$preferably

$0.5 \leq \frac{G^{\prime}}{\left( {G^{''} + G^{\prime}} \right)} \leq {0.98.}$

The product according to the invention is manufactured in predefinedphysical forms, or structures, such as a particle, a fibre, a string, astrand, a net, a film, a disc or a bead, which optionally is hollow orconsists of different layers of hyaluronic acid materials. Structuresthat are longitudinally extended, or rod-shaped, and have a ratiobetween their longitudinal extension and their largest lateral extensionof 5:1 or higher, such as 10:1 or higher, e.g. 20:1 or higher, andoptionally 100 000:1 or lower, such as 25 000:1 or lower, e.g. 100:1 orlower, are particularly useful as medical or cosmetic implants, becausethey can be dimensioned to avoid migration by having a sufficientlength, and can at the same time readily be administered by injectionthrough a needle due to the limited width. When a fibre, string orstrand is formed, it is preferred that the ratio between its length andits width or its average diameter, is 5:1 or higher, such as 10:1 orhigher, e.g. 20:1 or higher, and optionally 100 000:1 or lower, such as25 000:1 or lower, e.g. 100:1 or lower. By way of example, across-linked hyaluronic acid product according to the invention with asingle strand or fibre shape filling up a 20 mL syringe may have athickness of 1 mm and a length of 25 m in swelled state, i.e. a ratiobetween its longitudinal extension and its largest lateral extension of25000:1. A preferred string thickness interval is 50-200 μm.

The gel products may be designed as hollow containers that contain humancells, drugs or other substances. According to one embodiment of theinvention, a cross-linked HA gel product may be useful as a drugdelivery device and be used in a method of drug delivery. According toan embodiment of the invention, a cross-linked HA gel product may becombined with non-cross-linked HA or cross-linked HA with a differentdegree of modification or degree of cross-linking. For instance, thecross-linked gel product may be produced in a desired container shape.The container may form a reservoir for non-cross-linked HA orcross-linked HA with a different, e.g. lower, degree of modification,which can then be slowly released or kept contained for the purpose ofmodulating the total strength of the resulting combination product.

Due to the cross-linking, the shaped hyaluronic acid product is acontinuous network of interconnected and entangled hyaluronic acidchains which under non-precipitating conditions absorbs liquid (swells)and forms a gel. The swelling can be allowed to proceed until the gel isfully swollen and no further liquid can be absorbed. Thus, the shapedcross-linked hyaluronic acid product may be provided in fully swollenstate. The swelling can also be interrupted at an earlier stage toobtain a partially swollen gel. A partially swollen gel can be useful asan intermediate for further processing of the gel, for instanceproduction of gel structures or pieces of a desired size and shape. Itmay also be convenient to use a partially swollen gel duringimplantation thereof at a desired site to facilitate administration andminimize patient discomfort. The shaped cross-linked hyaluronic acidproduct according to the invention can therefore also be provided inpartially swollen or non-swollen state.

The shaped cross-linked hyaluronic acid product according to theinvention is useful in cosmetic or medical surgery. Non-limitingexamples of cosmetic surgery are dermal filling and body contouring.Non-limiting examples of medical surgery are dermal filling, bodycontouring, prevention of tissue adhesion, orthopaedic applications,incontinence treatment, treatment of vesicoureteral reflux (VUR), andformation of channels for draining purposes, e.g. in ophthalmology, andfor keeping tissues apart. The shaped cross-linked hyaluronic acidproduct according to the invention is also useful in drug delivery. Itcan furthermore be used as a film for post-surgical (interperitorial)adhesion and in hip and joint therapy.

According to one aspect, the present invention provides a method oftreatment of a subject undergoing cosmetic or medical surgery, involvingadministration of a shaped cross-linked hyaluronic acid productaccording to the invention to a subject in need thereof. Non-limitingexamples of medical surgery are dermal filling, body contouring,prevention of tissue adhesion, orthopaedic applications, e.g. hip andjoint therapy, and formation of channels for draining purposes, e.g. inophthalmology, and for keeping tissues apart

According to one embodiment of the invention, the shaped cross-linkedhyaluronic acid gel product can be brought into further structures orpieces with different shapes having a size, when subjected to aphysiological salt solution, above 0.1 mm. It is preferred thatcross-linked hyaluronic acid products according to the invention have alongitudinally extended shape, and that the largest lateral extension isless than 5 mm, preferably less than 1.5 mm, such as less than 0.8 mm oreven less than 0.5 mm in fully swollen state. This is advantageous forthe purpose of injecting the swollen gel products through a syringe ofdesired dimensions. It is also preferred that shaped cross-linkedhyaluronic acid products according to the invention have alongitudinally extended shape, and that the longitudinal extension ismore than 5 mm, preferably more than 500 mm (0.5 m), such as more than 5m or even more than 25 m in fully swollen state. Among manypossibilities, this longitudinal extension prevents migration and/ordisplacement of an implanted gel product in vivo.

The desired shape and size is arranged during the manufacturing of theproduct, i.e. by arranging the substrate in a desired shape prior tocross-linking. Another suitable way of obtaining a desired structuresize involves manufacturing a shaped cross-linked hyaluronic acid gel ata desired concentration and subjecting the gel to mechanical disruption,such as mincing, mashing or passing the swollen or partly swollen gel,or the precipitated cross-linked product, through a filter or mesh withsuitable pore size. The resulting gel particles or pieces are dispersedin a physiological salt solution, resulting in a gel dispersion orslurry with particles of desired size and shape. Depending on the shape,the size of a gel structure may be determined in any suitable way, suchas by laser diffraction, microscopy, filtration, etc, and is decided bythe longest distance between two ends of the particle. For sphericalstructures, the diameter equals the size for this purpose.

Useful gel structure size ranges and shapes depend on the intendedapplication. For soft tissue augmentation, preferably subcutaneousadministration, submuscular administration or supraperiostaladministration, gel particles, pieces or fibres having a size, whensubjected to a physiological salt solution, of more than 0.1 mm areuseful. The term “soft tissue augmentation”, as used herein, refers toany type of volume augmentation of soft tissues, including, but notlimited to, facial contouring (e.g. more pronounced cheeks or chin),correction of concave deformities (e.g. post-traumatic, HIV associatedlipoatrophy) and correction of deep age-related facial folds. Thus, softtissue augmentation may be used solely for cosmetic purposes or formedical purposes, such as following trauma or degenerative disease.These two purposes are easily distinguished by the skilled person. Theterm “soft tissue”, as used herein, refers to tissues that connect,support, or surround other structures and organs of the body. Softtissue includes muscles, fibrous tissues and fat. Soft tissueaugmentation may be performed in any mammal, including man. It ispreferred that the method is performed in a human subject.

The terms “subepidermal administration” or “subcuticularadministration”, as used herein, refer to administration beneath theepidermis of the skin, including administration into the dermis,subcutis or deeper, such as submuscularly or into the periosteum whereapplicable (in the vicinity of bone tissue).

Administration of gel structures may be performed in any suitable way,such as via injection from standard cannulae and needles of appropriatesizes or surgical insertion, e.g. in the case of administration of afilm. The administration is performed where the soft tissue augmentationis desired, such as the chin, cheeks or elsewhere in the face or body.

An implant according to the invention may be an aqueous compositioncomprising the shaped cross-linked hyaluronic acid product according tothe invention, e.g. in the shape of ≥0.1 mm large hyaluronic acid gelstructures, such as particles, beads, fibres or cut-out stripes, andoptionally a buffering agent. The composition may typically contain aphysiological salt buffer. The composition may further comprise othersuitable additives, such as local anaesthetics (e.g. lidocainehydrochloride), anti-inflammatory drugs, antibiotics and other suitablesupportive medications, e.g. bone growth factors or cells. The shapedcross-linked hyaluronic acid product according to the invention, or anaqueous composition thereof, may be provided in a pre-filled syringe,i.e. a syringe that is pre-filled with a sterilized, shaped cross-linkedhyaluronic acid product or a sterilized aqueous composition comprisingthe shaped product. Optionally, the shaped cross-linked hyaluronic acidproduct may be kept in precipitated form in a syringe, bag or othersuitable container and be brought to its non-precipitated form prior toinjection or in the body following injection thereof.

It is preferred that the swelled or partly swelled, shaped cross-linkedhyaluronic acid product is autoclavable, since this is the mostconvenient way of sterilising the final product. This allows forpreparation of a sterile, shaped cross-linked hyaluronic acid product.

It goes without saying that the size of the gel structures, e.g. fibres,according to the invention is dependent upon how much the gel has beenallowed to swell, and the ionic strength of the buffer, solution orcarrier that is included in and/or surrounding the gel structures.Throughout this specification, given structure sizes assumephysiological conditions, particularly isotonic conditions. It shall benoted that, while it is preferred that the gel structures contain andare dispersed in a physiological salt solution, it is contemplated thatthe gel structures according to the invention can temporarily be broughtto different sizes by subjecting the gel structures to a solution ofanother tonicity, different pH or if the gel structures have not beenallowed to swell to their maximum size.

As used herein, a physiological, or isotonic, solution is a solutionhaving an osmolarity in the range of 200-400 mOsm/l, preferably 250-350mOsm/I, more preferably approximately 300 mOsm/l. For practicalpurposes, this osmolarity is easily achieved by preparation of a 0.9%(0.154 M) NaCl solution.

The shaped cross-linked hyaluronic acid gel product according to theinvention is stable, but not permanent, under physiological conditions.The stability in vitro is demonstrated in Example 7 by an acceleratedstability study at 60° C. for 14 days. According to an embodiment of theinvention, at least 70%, preferably at least 90%, of the shapedcross-linked hyaluronic acid gel product remains for at least two weeksin vivo, more preferably between two weeks and two years. The term“degraded” implies that less than 20%, preferably less than 10%, of themedium remains in the body.

The shaped cross-linked hyaluronic acid gel product according to theinvention is more resistant to biodegradation in vivo than naturalhyaluronic acid. The prolonged presence of the stable gel product isadvantageous for the patient, since the time between treatments isincreased. It is also important that the product is highly similar tonative hyaluronic acid, in order to maintain the high biocompatibilityof the native hyaluronic acid. A product that is similar to native HAshould be degradable by the enzyme hyaluronidase, preferably to a degreeof at least 99%. The biodegradability of the firm hyaluronic acid gelproducts according to the invention are demonstrated in Example 8, wherethey are recognized by hyaluronidase. This reflects their similarity tonative hyaluronic acid.

Definitions

Throughout this disclosure, the terms below are defined as follows.

Term Property Meaning HA HA refers to sodium hyaluronate Gel-formGel-form HA is the cross-linked HA that HA cannot be extracted from thegel by rinsing with e.g. saline, as opposed to the extractable HAExtractable Extractable HA is the HA, cross-linked or HA notcross-linked, that can be extracted by rinsing with e.g. saline C_(HA)HA concentration$C_{HA} = {{\frac{m_{HA}}{m_{sample}}\mspace{14mu}{or}\mspace{14mu} C_{HA}} = \frac{m_{HA}}{v_{sample}}}$Expressed in mg/g, mg/mL, %(w/w), %(w/v) SwD Swelling Degree${SwD} = \frac{m_{{fully}\mspace{14mu}{swollen}\mspace{14mu}{gel}}}{m_{{gel} - {{form}\mspace{14mu}{HA}\mspace{14mu}{in}\mspace{14mu}{fully}\mspace{14mu}{swollen}\mspace{14mu}{gel}}}}$SwD is preferably expressed in g/g, mL/g, or as a dimensionless number.SwD is the inverted concentration of gel-form HA in a gel that is fullyswollen in 0.9% saline, i.e. the volume, or mass, of fully swollen gelthat can be formed per gram dry cross-linked HA. SwD describes themaximum liquid-absorbing (0.9% saline) capability of the product.C_(min) Minimum HA Concentration of gel-form HA in a gel that isConcentration fully swollen in 0.9% saline, normally expressed in mg/gor mg/mL. C_(min) ⁻¹ = SwD GelC Gel Content${GelC} = \frac{m_{{{HA}\mspace{14mu}{in}\mspace{14mu}{gel}} - {form}}}{m_{{HA}\mspace{14mu}{total}}}$Expressed as g/g, a dimensionless number, or %. The Gel Content is theproportion of HA that is bound in gel-form out of the total HA contentin the product. MoD Degree of Modification${MoD} = \frac{n_{{bound}\mspace{14mu}{crosslinking}\mspace{14mu}{agent}}}{n_{{disaccharide}\mspace{14mu}{units}}}$Expressed as mole/mole, a dimensionless number, or mole %. MoD describesthe amount of cross-linking agent(s) that is bound to HA, i.e. molaramount of bound cross-linking agent(s) relative to the total molaramount of repeating HA disaccharide units. MoD reflects to what degreethe HA has been chemically modified by cross-linking agent(s). CrRCross-linker ratio${CrR} = \frac{n_{{HA} - X - {HA}}}{n_{{HA} - X - {HA}} + n_{{HA} - X}}$where X is a cross-linking agent. CrR can also be expressed as:${CrR} = \frac{\#{crosslinked}\mspace{14mu}{crosslinking}\mspace{14mu}{agents}}{\#{bound}\mspace{14mu}{crosslinking}\mspace{14mu}{agents}}$Expressed in mole/mole, a dimensionless number, or mole %. CrR describesthe proportion of total bound cross-linking agent (HA-X-HA and HA-X)that has bound two (or more) disaccharides (only HA-X-HA). MoEModification Efficiency ${MoE} = \frac{C_{\min}}{MoD}$ MoE is adimensionless number obtained as the ratio between C_(min) expressed inmg/g and MoD expressed in %. MoE describes the amount ofinterconnections, caused both by chemical modification and frommolecular entanglements, which have been achieved at the cost of acertain degree of chemical modification by cross-linking agent(s).Example: MoE for a product with C_(min) = 35 mg/mL and MoD = 1.15% isapproximately 30, and is calculated as follows:${MoE} = {\frac{35}{1.15} \approx 30}$ G′ Elastic The elastic modulusdescribes the resistance modulus of the gel to elastic deformation, andis expressed in Pa (Pascal). A strong gel will give a larger numbercompared to a weak gel. G″ Viscous The viscous modulus describes theresistance modulus of the gel to viscous deformation, and is expressedin Pa (Pascal). Together with G′, it describes the total resistance todeformation.

Without desiring to be limited thereto, the present invention will inthe following be illustrated by way of examples.

Examples Section

Analytical Methods

Determination of HA Concentration

The method for determination of HA content is adopted from the assaytest for sodium hyaluronate described in Ph. Eur. 1472. The principlefor the method is that a condensation reaction of the furfuralderivatives formed by heating in sulphuric acid occurs with thecarbazole reagent, forming a purpur colored product. The reaction isspecific for the D-glucuronic acid part of HA. The absorbance ismeasured at 530 nm and glucuronic acid is used for standardization.

The product formed from the content of D-glucuronic acid (GlcA) in thesample is determined by reaction with carbazole. To get homogeneoussample solutions, the stabilized gel of HA is degraded with sulphuricacid at 70° C. and diluted with 0.9% NaCl-solution. The solutions aremixed with sulphuric acid at 95° C. and thereafter with carbazolereagent. The reactions result in pink coloured solutions. The intensityof the colour is measured with a colorimeter at 530 nm, and theabsorbance of each sample is directly proportional to the GlcA-content.The HA content is calculated from the GlcA-content in each sample.

Determination of Gel Content (GelC)

GelC describes in % the proportion of the total HA that is bound in gelform. Gel content is defined as the amount of HA in a sample that doesnot pass through a 0.22 μm filter. GelC is calculated from the amount ofHA that is collected in the filtrate, here denoted extractable HA. Thegel content and the extractable HA content are given in percent of thetotal amount of HA in the gel sample. In short, the gel content isdetermined by mixing a certain amount of gel sample with 0.9% NaCl in atest tube. The gel is allowed to swell where after the NaCl-phase isseparated from the gel-phase by filtration through a 0.22 μm filter. Theconcentration of HA in the filtrate is determined according to theprocedure for determination of HA concentration.

Determination of Swelling Degree (SwD)

SwD describes the liquid-absorbing capability of a product, i.e. itscapability to absorb 0.9% NaCl. The product is typically a dry,cross-linked HA gel. The dry product may be precipitated or notprecipitated. SwD can be determined from the weight of fully swollenproduct that is formed upon swelling a certain weight of a dry productin saline. If the dry product is precipitated, it will revert tonon-precipitated form under these conditions.

Dry product with a predetermined weight (typically 0.1 g) is subjectedto an excess of 0.9% NaCl(aq), and the product is allowed to swell forone hour at room temperature (23° C.). The fully swollen products arecollected and weighed after removing non-absorbed liquid. Removal ofnon-absorbed liquid also removes any substances that are not bound to orentangled in the cross-linked gel, such as non-cross-linked and weaklycross-linked hyaluronate molecules. Since these which will notcontribute to the weight of the fully swollen product, thereby giving anapparently lower SwD, correction for the gel content should be made inorder to determine the true SwD. If the gel content is considered closeenough to 100% that the result is not affected, this correction may beomitted.

SwD is calculated as the ratio between weight of product, fully swollenand rinsed from extractable HA as described above, and weight of the drycross-linked HA in the product:

${{SwD} = {\frac{m_{{fully}\mspace{14mu}{swollen}\mspace{14mu}{gel}}}{m_{{gel} - {{form}\mspace{14mu}{HA}\mspace{14mu} i\; n\mspace{14mu}{fully}\mspace{14mu}{swollen}\mspace{14mu}{gel}}}} = \frac{m_{{fully}\mspace{14mu}{swollen}\mspace{14mu}{gel}}}{m_{dry} \times {GelC}}}},$wherein GelC is expressed as a dimensionless number. If GelC isconsidered close to 1 (100%), SwD can be calculated as:

${SwD} = \frac{m_{{fully}\mspace{14mu}{swollen}\mspace{14mu}{gel}}}{m_{dry}}$

Three techniques of collecting the fully swollen product and removingnon-absorbed liquid have been used: 1) collecting the swollen productpiece by piece, allowing the products to briefly touch a dry surface, 2)collecting all of the swollen product using a metal net, allowing thenet to briefly touch a dry surface, 3) removing liquid from the swollenproduct by suction through a 0.2 μm filter. In the latter case, theweight of the fully swollen product was not weighed directly, butcalculated from the weight of the dry sample before swelling, the weightof the added liquid and the weight of the liquid removed uponfiltration, using the formula:m _(fully swollen gell product) =m _(added liquid) −m _(removed liquid)+m _(dry product)

Notably, a stronger gel will have a lower SwD, while a weaker gel willhave a higher SwD.

Determination of Minimum Concentration (C_(min))

C_(min) describes the concentration of gel-form HA in a cross-linked HAgel product, fully swollen in 0.9% NaCl after all extractable HA isremoved. Since the product cannot absorb more liquid, this concentrationis the minimum HA concentration that can be obtained for this particulargel product. Notably, a stronger gel will have a higher C_(min), while aweaker gel will have a lower C_(min).

The C_(min) is determined in analogy with the determination of SwD asset out above, using the relation:

$C_{\min} = {\frac{1}{SwD}.}$Determination of Degree of Modification (MoD)

MoD describes the molar amount of bound cross-linking agent(s) relativeto the total number of repeating HA disaccharide units. This measuredoes not distinguish between mono-linked and actually cross-linkedcross-linking agent(s), i.e. all cross-linking agent(s) that is bound toHA via at least one covalent bond is included. For instance, a MoD of 1%for a HA gel cross-linked with BDDE means that there is 1 bound(monolinked or cross-linked) molecule of BDDE per 100 disaccharide unitsin the HA gel.

MoD is determined using NMR spectroscopy on enzymatically degraded gelproduct. Soluble HA, residual (non-bound) cross-linking agent(s) andderivatives thereof are washed away prior to the degradation of the gelby filtration on a 0.22 μm filter. The gel product is degraded at 37° C.by enzymatic treatment using Chondroitinase AC from Arthrobacteraurescens. The degraded gel product is subjected to NMR spectroscopy byrecording one-dimensional ¹H NMR spectra on a 400 MHz spectrometer,equipped with a standard 5 mm probe.

The NMR spectra are evaluated by integration of the signal at δ_(H) 1.6ppm, which origins from four protons in the linked BDDE molecule, andthe signals at δH 2.0 ppm, which is from the three protons in the CH₃groups on the N-acetylglucosamine residues of the HA disaccharides. Theratio between the integrals for these two signals is proportional to theratio between the molar amount of bound BDDE and disaccharides aftercorrection for the number of protons responsible for each signal, hencegiving MoD.

${MoD} = \frac{n_{{bound}\mspace{14mu}{crosslinking}\mspace{14mu}{agent}}}{n_{{disaccharide}\mspace{14mu}{units}}}$Determination of Modification Efficiency (MoE)

MoE is the ratio between the minimum HA concentration and the degree ofmodification of a gel, i.e.:

${MoE} = {\frac{C_{\min}}{MoD} = \frac{1}{{SwD} \times {MoD}}}$

C_(min) (mg/g or mg/mL) and MoD (%) are determined as describedpreviously. Since C_(min) is closely related to the strength of a gel,MoE is a measure of how efficient the cross-linking procedure is inproducing a gel of desired strength. A process with a high MoE willproduce a gel with a high C_(min) and a low MoD, i.e. a strong gel isproduced despite limited chemical modification of the HA.

Determination of Cross-Linker Ratio (CrR)

CrR describes the proportion of total bound cross-linking agent(s)(HA-X-HA and HA-X) that has bound two (or more) disaccharides (onlyHA-X-HA):

${CrR} = \frac{n_{{HA} - X - {HA}}}{n_{{HA} - X - {HA}} + n_{{HA} - X}}$where X is a cross-linking agent.

The method is based on determination of HA-X fragments using SEC-UV-MSfollowing degradation of the HA gel using chondroitinase AC fromArthrobacter aurescens or chondroitinase ABC from Proteus vulgaris intofragments consisting of the main disaccharide (Δdi-HA) and fragmentswith bound cross-linking agent (HA-X) containing 1-16 disaccharides. Thefragments are separated using size exclusion chromatography (SEC), anddetected using mass spectrometry (MS). The peak areas for each group offragment are summed, and CrR is calculated as:

${CrR} = \frac{{Peak}\mspace{14mu}{area}_{{HA} - X - {HA}}}{{{Peak}\mspace{14mu}{area}_{{HA} - X - {HA}}} + {{Peak}\mspace{14mu}{area}_{{HA} - X}}}$

It is assumed that all types of HA-X fragments have the same response inthe MS detector, i.e. a certain peak area corresponds to a given molaramount for all types of HA-X fragments (Kenne et al., CarbohydratePolymers 91 (2013) 410-418).

When determining CrR, care should be taken to only include BDDE bound byether linkages. Depending on the conditions during cross-linking, BDDEcan bind to the HA via both ether and ester linkages. Since the esterlinkages are easily hydrolyzed, it is only the ether-bound BDDE thatwill contribute to the gel strength and duration in the long term. Thefragments with ester-bound BDDE have the same mass as the ether-boundBDDE but can be detected as they have slightly different chromatographicretention times. To determine the CrR without any ester-bound BDDE, thesamples should be hydrolyzed before analysis. Hydrolysis of the samplescould e.g. be made by adding base and/or heat before or after theenzymatic degradation.

Determination of pH

The pH determination is performed potentiometrically at ambienttemperature using a glass electrode. The method used for pHdetermination of the swelled product is based on USP method <791>.Procedure: Calibrate the pH-meter at ambient room temperature withbuffer solutions for standardization at pH 7.0 and 4.0. Transfer about1.2 mL of sample (for every measurement) into a suitable container. Makesure that the sample is at room temperature. Measure the pH onduplicates of the sample. Wash the electrode in distilled water and wipeoff carefully between each measurement.

The method for pH determination of the process solutions, notably theorganic solvent is as above, but with calibration at pH 7.0 and 10.0.

The pH values measured in a mixture of water and organic solvent(s)differ compared to pH measured in pure water solutions for the sameconcentration of base, e.g. sodium hydroxide (NaOH). Therefore, theexpression apparent pH (pH_(app)) is used for pH measured in an aqueousmixture of organic solvent(s). The apparent pH is depending on severalfactors, including the type of organic solvent(s), the concentration oforganic solvent(s), the temperature, the ion strength and the presenceof other compounds in the mixture.

The apparent pH values reported in the experimental section weremeasured with a Mettler Toledo MA 234 pH/Ion analyzer with a MettlerToledo Inlab Routine Pro electrode for pH range 0-14.

Rheometry

Rhemometry in the oscillating mode is used to determine the viscoelasticproperties of the swelled gel product. The elastic modulus (G′)describes the gel strength in terms of the gels physical resistance toelastic deformation. The viscous modulus (G″) describes the gel strengthin terms of the gels physical resistance to viscous deformation. Themeasurement is performed using an oscillating rheometer.

Rheometry measurements are performed as follows. Frequency sweeps aremade with a resting time of at least 15 minutes between sample loadingand measurement, and a strain (γ) of 0.1%. A parallel plate probe with adiameter of 25 mm is used with a gap of 2 mm. Average values of theelastic modulus (G′) and viscous modulus (G″) are evaluated at 0.1 and 1Hz from the frequency sweeps. Amplitude sweeps are made at 1 Hz toverify that the frequency sweep was performed at a strain (γ) within thelinear viscoelastic range.

Determination of Enzyme Degradability

Enzyme degradation is performed in order to verify that the cross-linkedgel is equal to native HA regarding biodegradability by confirming thatit is degradable by hyaluronidase from sheep testes.

The product (dry, or swelled to a known concentration) with apredetermined weight is allowed to swell over night at 37° C. in 100 mLof 0.9% saline. Hyaluronidase from sheep testes (Type II, H2126 Sigma)is added to obtain an enzyme concentration of 200 U/mL, and thepreparation is set to shake over night at 37° C. The solution isfiltrated through a 0.22 μm filter, and the amount of HA in the filtrateis determined using the carbazole method. The degradation is calculatedas the ratio of the amount of HA in the filtrate to the total amount ofHA in the sample.

EXAMPLES Example 1 Cross-linking Procedures

Approximately 1 g sodium hyaluronate having a molecular weight of 0.8-3MDa was dissolved in 10-50 mL of an aqueous solution containing 0-40%ethanol, and 0-120 mM strong base. After complete dissolution, theresulting hyaluronate solution had a pH_(app) of 6-13, measured asdescribed above. (Preparation step)

The hyaluronate solution was extruded through an opening with a 0.3-0.5mm diameter into a liquid medium containing an organic solvent. Theextruded hyaluronate solution immediately precipitated into hyaluronatefibres. (Precipitation step)

Approximately 1 g of precipitated HA fibres was then transferred toapproximately 20-70 mL of an aqueous cross-linking medium containing65-80% of an organic solvent, 0.3-1.2 g (30-230 mM) cross-linking agent,and 1-75 mM strong base. The hyaluronate remained precipitated and inthe form of fibres. The resulting cross-linking medium including HA hada pH above 11.5. The HA fibres were allowed to cross-link in theaqueous/organic cross-linking medium. (Cross-linking step)

The thus obtained cross-linked hyaluronate fibres were then removed fromthe cross-linking medium and neutralized with phosphoric acid or thelike. The cross-linked HA fibres were dried in vacuum chamber underreduced pressure (˜200 mbar) at room temperature for approximately 1hour.

The precipitated intermediate was characterised as follows. The weightof the dry cross-linked hyaluronate fibres was determined. The driedfibres were soaked in an excess of 0.9% aqueous saline and allowed toabsorb liquid freely. The fibres then formed an aqueous gel, and salinewas absorbed until equilibrium (maximum) swelling of the fibres wasobtained. The gel fibres were removed manually from the solution ofexcess saline and non-cross-linked hyaluronate and transferred directlyto a balance. The weight of the fully swollen fibres was determined.From the saline uptake results, a degree of swelling (SwD) wasdetermined, corresponding to a certain sodium hyaluronate concentrationin fully swelled gel (C_(min)).

The degree of modification (MoD) of the resulting cross-linkedhyaluronate fibre was determined by NMR spectroscopy as described above.Briefly, the intensity for the signal from linked BDDE is related to thesignal from the acetyl group in HA. The molar ratio of linked BDDErelative HA can then be calculated after correction for the number ofprotons giving rise to the signals. The corresponding modificationefficiency (MoE) was calculated from the swelling degree.

The described precipitated intermediates of the cross-linked HA fibreswere allowed to swell to a defined HA-concentration as follows.Phosphate buffered saline solution (4 mM phosphate and 150 mM NaCl) orthe like was added to the dried HA fibres to a concentration of about10-40 mg HA/mL. The partly swollen gel was filled into 10 mL plasticsyringes. The filled syringes were sterilized using moist heat at 123°C. with a final equivalent time (F₀) of approximately 20 minutes. Thecontent of the autoclaved syringes were characterised regarding pH, HAconcentration, degree of swelling (SwD), and minimum concentration(C_(min)) with the methods described above, and MoE was calculated fromthe MoD determined for the intermediate.

Cross-linked HA fibres were prepared as described above. Analytical datafor precipitated intermediates are reported in the Table 1, and dataobtained for swelled and autoclaved products are shown in Table 2.

TABLE 1 Characterisation of product (precipitated intermediate) AmountExp Amount dry swollen SwD* C_(min) MoD Id HA (mg) HA (mg) (g/g) (mg/mL)(%) MoE 1 — — — — 0.3 — 2 23 1181 51 20 0.7 28 3 15 2200 147   7 0.2 384 17 1070 62 16 0.2 73 5 17 1505 89 11 0.4 30 6 16 2110 133   8 0.2 42 7— — — — 1.3 — 8 — — — — 0.4 — 9 — — — — 0.5 — 10 — — — — 0.2 — 11 — — —— 0.3 — 12 16  501 32 31 1.7 19 13 15  313 21 48 0.3 190  14   19.8  63232 31 0.7 46 15 — — — — 0.5 — *Measured according to procedure 1

TABLE 2 Characterisation of swelled and autoclaved product HA Expconcentration SwD** C_(min) Id pH (mg/mL) (mL/g) (mg/mL) MoE  1 6.9 20*110 9 28  2 — — — — —  3 7.3 19  85 12 65  4 — 20* — — —  5 — — — — — 6*** 7.2 20* 139 7 40  7 7.6 20* 23 43 32  8 6.6 20* 40 25 59  9 7.120* 21 47 97 10 7.2 20* 96 10 44 11 7.3 20* 55 18 55 12 — — — — — 13 7.320* 23 44 174 14 — — — — — 15 7.9 20* 97 10 21 *estimated HAconcentration **measured according to procedure 3 ***2 mg lidocainehydrochloride was added per mL phosphate buffer saline solution in theswelling step described above

Example 2 Cross-linking with Different Cross-linking Agents

A hyaluronate solution was prepared as set out in Example 1 Aftercomplete dissolution, the resulting hyaluronate solution had a pH above9, measured as described above. The hyaluronate solution was extrudedthrough an opening with a 0.5 mm diameter into a liquid medium of 99.5%ethanol. The extruded hyaluronate solution immediately precipitated intohyaluronate fibres.

Approximately 1 g of precipitated HA fibres was transferred to anaqueous/organic cross-linking medium containing 70% ethanol,cross-linker and NaOH. The hyaluronate remained precipitated and in theform of fibres. The resulting cross-linking medium including HA, had apH above 11.5.

The HA fibres were allowed to cross-link in the aqueous/organiccross-linking medium. The thus obtained cross-linked hyaluronate fibreswere then removed from the cross-linking medium and neutralized withphosphoric acid. The cross-linked HA fibres were dried in a vacuumchamber under reduced pressure (˜200 mbar) at room temperature forapproximately 2 hours.

The described precipitated intermediate of the cross-linked HA fibreswas allowed to swell to a defined HA concentration as follows. Phosphatebuffered saline solution was added to the dried HA fibres to aconcentration of about 20 mg HA/mL. The partly swollen gel was filledinto 10 mL plastic syringes. The filled syringes were sterilized usingmoist heat at 123° C. with a final F₀ of approximately 20 minutes. Thecontent of the autoclaved syringes was characterised regarding pH,degree of swelling (SwD procedure 3), minimum concentration (C_(min)),degree of modification (MoD) and modification efficiency (MoE), with themethods described above. The results shown in Table 3 imply that thedifferent diepoxides are useful cross-linking agents in the describedprocess.

TABLE 3 Characterisation of product SwD* C_(min) MoD Cross-linker pH(mL/g) (mg/mL) (%) MoE EDDE** 7.6 24 41 1.5 27 EDDE** 7.3 12 86 2.8 31Diepoxyoctane 7.9 97 10 0.5 21 *Measured according to procedure 3, HAconc assumed to be 20 mg/mL **1,2-ethanediol diglycidyl ether, purity50%

Example 3 Cross-linking in Methanol

A hyaluronate solution was prepared as set out in Example 1. Aftercomplete dissolution, the resulting hyaluronate solution had a pH above9, measured as described above. The hyaluronate solution was extrudedthrough an opening with a 0.5 mm diameter into a liquid medium of 99.5%ethanol. The extruded hyaluronate solution immediately precipitated intohyaluronate fibres.

Approximately 0.75 g of precipitated HA fibres was then transferred toan aqueous cross-linking medium containing 80% methanol, 1,4-butanedioldiglycidyl ether (BDDE), and NaOH. The hyaluronate remained precipitatedand in the form of fibres. The resulting cross-linking medium includingHA, had a pH above 11.5.

The HA fibres were allowed to cross-link in the aqueous/organiccross-linking medium. The thus obtained cross-linked hyaluronate fibreswere then removed from the cross-linking medium and neutralized withphosphoric acid. The cross-linked HA fibres were dried in vacuum chamberunder reduced pressure (˜200 mbar) at room temperature for approximately1 hour.

The degree of swelling was measured according to SwD procedure 1. Thedried intermediate of the cross-linked fibres were allowed to swell to20 mg HA/mL in a phosphate buffered saline solution. The swollen gel wasfilled into 10 mL plastic syringes. The filled syringes were sterilizedusing moist heat at 123° C. with a final F₀ of approximately 20 minutes.The content of the autoclaved syringes were characterised with respectto pH and degree of modification (MoD) according to methods describedabove. The resulting gel formulation had a pH of 6.8, and a MoD of0.34%.

Example 4 Cross-linking in Isopropanol

Hyaluronate solution was prepared as described in Example 3.Approximately 0.5 g of precipitated HA fibres was then transferred to 30mL of an aqueous cross-linking medium containing 70% isopropanol,1,4-butanediol diglycidyl ether (BDDE), and NaOH. The hyaluronateremained precipitated and in the form of fibres. The resultingcross-linking medium including HA, had a pH above 11.5.

The HA fibres were allowed to cross-link in the aqueous/organiccross-linking medium. The thus obtained cross-linked hyaluronate fibreswere removed from the cross-linking medium and neutralized withphosphoric acid. The cross-linked HA fibres were then dried in vacuumchamber under reduced pressure (˜200 mbar) at room temperature forapproximately 1 hour.

The dried intermediate of the cross-linked fibres were allowed to swellin phosphate buffered saline solution to a HA-concentration of 20 mgHA/mL. The swollen gel was filled into 10 mL plastic syringes. Thefilled syringes were sterilized using moist heat at 123° C. with a finalF₀ of approximately 20 minutes. The content of the autoclaved syringeswere characterised with respect to pH, degree of swelling (according toprocedure 3), gel content and degree of modification (MoD) according tomethods described above. The minimum concentration C_(min) and themodification efficiency (MoE) were calculated as described above. Theresulting gel formulation had a pH of 7.6, SwD 47 g/g HA, C_(min) 21mg/mL, MoD 0.41%, and MoE 52.

Example 5 Cross-linking in Desired Shapes and Structures

A hyaluronate solution was prepared as set out in Example 1. Aftercomplete dissolution, the resulting hyaluronate solution had a pH above9, measured as described above.

Precipitated HA substrates with different shapes, or structures, wereprepared from the above hyaluronate solution by:

-   1) extrusion through an opening with a diameter of 0.5 mm forming    droplets;-   2) extrusion through an opening with a diameter of 0.5 mm forming a    net; and-   3) spreading on a plastic foil forming a film, and subsequently    immersed into a liquid medium of 99.5% ethanol. The extruded    hyaluronate solution immediately precipitated into hyaluronate    droplets, net and film, respectively.

Approximately 1 g of each precipitated HA substrate was then transferredto 45 mL of an aqueous cross-linking medium containing 70% ethanol,1,4-butanediol diglycidyl ether (BDDE), and NaOH. During thecross-linking reaction, the hyaluronate remained precipitated and in thepredetermined forms. The resulting cross-linking medium including HA,had a pH above 11.5.

After finished cross-linking, the cross-linked hyaluronate formulationswere removed manually from the cross-linking medium, neutralized withphosphoric acid and dried in vacuum chamber under reduced pressure (˜200mbar) at room temperature for approximately 1 hour before they werecharacterised.

The degree of swelling (SwD) was determined for the droplets and thefilm. A known amount of the dry cross-linked hyaluronate sample soakedin an excess of 0.9% aqueous saline and allowed to absorb liquid freely.The samples then formed aqueous gels, and saline was absorbed untilequilibrium (maximum) swelling of the samples was obtained. The gelsamples were then removed manually from the solution of excess salineand eventually non-cross-linked residual hyaluronate, and weretransferred directly to a balance. The maximum saline uptake wasestimated by comparing the weight for the swollen gels with the initialweight of the dry material. The results are presented in Table 4 both asdegree of swelling (SwD procedure 1) and minimum concentration(C_(min)). The net was allowed to swell in an excess of 0.9% aqueoussaline as described above. The swelled gel net is shown in FIG. 1.

The degree of modification (MoD) of the cross-linked hyaluronatedroplets and film was determined by NMR spectroscopy as described above.Briefly, the intensity for the signal from linked BDDE is related to thesignal from the acetyl group in HA. The molar ratio of linked BDDErelative HA was then calculated after correction for the number ofprotons giving rise to the signals. The results from MoD and thecalculated modification efficiency (MoE) are presented in Table 4.

The dried intermediate of the cross-linked droplets were allowed toswell in phosphate buffered saline solution to a HA concentration of 20mg HA/mL. The swollen gel was filled into 10 mL plastic syringes. Thefilled syringes were sterilized using moist heat at 123° C. with a F₀ ofapproximately 20 minutes. The results from the characterisation ofautoclaved droplets are also presented in Table 4.

TABLE 4 SwD C_(min) MoD Sample (g/g HA) (mg/mL) (%) MoE Droplets  60* 170.47 36 Autoclaved  58** 17 0.47 37 droplets Film 115* 9 0.43 20*measured according to procedure 1 **measured according to procedure 3

The results show that it is possible to produce cross-linked HAmaterials with a predefined form that is retained during thecross-linking process and autoclaving.

Example 6 Cross-linking of Alkaline Fibres

A hyaluronate solution was prepared as set out in Example 1. Aftercomplete dissolution, the resulting hyaluronate solution had a pH above9, measured as described above. The hyaluronate solution was extrudedthrough a 21 G needle (inner diameter 0.5 mm) into a liquid medium of99.5% ethanol. The extruded hyaluronate solution immediatelyprecipitated into hyaluronate fibres.

Approximately 1 g of precipitated, unwashed HA fibres was thentransferred to 68 mL of an aqueous cross-linking medium containing 70%ethanol and 1,4-butanediol diglycidyl ether (BDDE). The hyaluronateremained precipitated, alkaline and in the form of fibres. The resultingcross-linking medium after addition of the alkaline HA fibres, had a pHabove 11.5.

The HA fibres were allowed to cross-link in the aqueous/organiccross-linking medium. The thus obtained cross-linked hyaluronate fibreswere then removed from the aqueous cross-linking medium, neutralizedwith phosphoric acid, dried in vacuum chamber under reduced pressure(˜200 mbar) at room temperature for approximately 1 hour andcharacterised.

The dried intermediate of the cross-linked fibres were allowed to swellto a HA-concentration of approximately 20 mg/mL in phosphate bufferedsaline solution. The partly swollen gel was filled into 10 mL plasticsyringes. The filled syringes were sterilized using moist heat at 123°C. with a final F₀ of approximately 20 minutes. The content of theautoclaved syringes were characterised with respect to pH, degree ofswelling (SwD), gel content and degree of modification (MoD) accordingto methods described above. The minimum concentration C_(min) and themodification efficiency (MoE) were calculated as described above. Theresults are presented in Table 5.

TABLE 5 pH gel SwD* C_(min) MoD Formulation formulation (g/g HA) (mg/mL)(%) MoE 1 6.6 170 6 0.10 59 2 6.8 51 20 0.25 78 *Measured according toprocedure 3 and corrected for gel content

Thus, cross-linking of alkaline fibres in neutral cross-linking mediumcontaining 70% alcohol with a final apparent pH above 11.5 resulted inwell defined gel fibres.

Example 7 Biocompatibility and Stability Study

Four formulations were prepared as set out in Example 1 with the purposeto study the biocompatibility of the material in vivo. After completedissolution, the resulting hyaluronate solution had a pH above 9,measured as described above. The hyaluronate solution was extrudedthrough an 18 G (Ø 0.8 mm; formulations 1 and 2) or 25 G (Ø 0.3 mm;formulations 3 and 4) wide opening into a liquid medium of 99.5%ethanol. The extruded hyaluronate solution immediately precipitated intohyaluronate fibres.

Approximately 1 g of precipitated HA fibres was then transferred to 60mL of an aqueous cross-linking medium containing 70% ethanol,1,4-butanediol diglycidyl ether (BDDE), and NaOH. Formulations 2 and 4were subjected to a four times higher BDDE concentration thanformulations 1 and 3. The hyaluronate remained precipitated and in theform of fibres. The resulting cross-linking medium including HA had a pHabove 11.5, as measured with the equipment described above.

The HA fibres were allowed to cross-link in the aqueous/organiccross-linking medium. The thus obtained cross-linked hyaluronate fibreswere then removed from the aqueous cross-linking medium, neutralizedwith phosphoric acid, dried in vacuum chamber under reduced pressure(˜200 mbar) at room temperature for approximately 1 hour andcharacterised.

The dried fibres were soaked in an excess of 0.9% aqueous saline andallowed to absorb liquid freely. The fibres then formed an aqueous gel,and saline was absorbed until equilibrium (maximum) swelling of thefibres was obtained. The gel fibres were then removed manually from thesolution of excess saline and non-cross-linked hyaluronate andtransferred directly to a balance. The maximum saline uptake wasestimated by comparing the initial weight of the precipitated materialwith the weight of the fully swollen fibres. The results obtained fromthe gel fibres before sterilization are presented in Table 6, both asdegree of swelling (SwD) and minimum HA concentration (C_(min)).

TABLE 6 Weight Weight of fully of dry swollen SwD* C_(min) Formu- fibrefibre (g/g (mg MoD CrR lation (mg) (mg) HA) HA/mL) (%) (%) MoE 1 14 114481 12 0.29 51 43 2 26 739 28 36 1.22 46 29 3 14 1397 100 10 0.28 50 36 414 478 34 29 1.13 47 26 *Measured according to SwD procedure 1

The degree of modification (MoD) of the resulting cross-linkedhyaluronate fibre was determined by NMR spectroscopy as described above.Briefly, the intensity for the signal from linked BDDE is related to thesignal from the acetyl group in HA. The molar ratio of linked BDDErelative HA can then be calculated after correction for the number ofprotons giving rise to the signals. FIG. 2A (formulation 1) and 2B(formulation 2) show 400 MHz ¹H NMR spectra of enzymatically degradedHA-gel in deuterated water (D₂O). Signals from linked BDDE and theN-acetyl group are marked in the spectra. The result of MoD and CrR, foreach respective formulation is presented in Table 6 together with themodification efficiency (MoE).

The dried intermediate of the cross-linked fibres were allowed to swellto a defined HA-concentration as follows. Phosphate buffered salinesolution was added to the dried HA-fibres to achieve an estimatedconcentration of approximately 20 mg HA/mL. Formulation 1 and 3 absorbedall the added solution, while formulation 2 and 4 did not. The swollengel was then filled under aseptic conditions into 1 mL glass syringes.The filled syringes were sterilized using moist heat at 125° C. with afinal F₀ of approximately 20 minutes.

The content of the autoclaved syringes were characterised with respectto HA concentration, gel content (GelC), degree of swelling (SwD), andpH according to methods described above. The results from the productafter sterilization are presented in Table 7.

TABLE 7 HA concentration SwD* Formulation (mg/mL) GelC (%) (g/g HA) pH 118   96 95 7.5 2 32 ~99 25 7.6 3 19   95 87 7.6 4 36 ~99 23 7.5*Measured according to SwD procedure 3 and corrected for gel content

It is apparent from Tables 6 and 7 that formulations 1 and 3 are lessrigid and less chemically modified than formulations 2 and 4. The highHA concentrations of formulations 2 and 4 reflect that the maximumuptake of water is low, and that it is not possible to achieve a gelproduct with lower HA concentration for a gel produced under theseconditions.

The formulations were analyzed regarding bioburden (pour plate method)and endotoxins (gel-clot method). All formulations had a bioburden of 0cfu/syringe and an endotoxin value of <0.21 EU/mL. The formulations allmet the requirements regarding purity for the biocompatibility study.

Microscopy

The swelled and autoclaved fibres were visualized by colouring thefibres in a Tolouidine blue water solution for 15 minutes. Themicroscopy images are shown in FIGS. 3A-D. 3A: Formulation 1; 3B:Formulation 2; 3C: Formulation 3; and 3D: Formulation 4. Bar=1 mm.

Stability Testing

Stability testing of the formulations was performed at 60° C./ambientrelative humidity (RH) for 14 days. The formulations all showed goodstability. FIG. 4 shows the increase of SwD over time at 60° C.(measured according to SwD procedure 3 and corrected for gel content).Formulation 1 (♦); Formulation 2 (▪); Formulation 3 (▴); and Formulation4 (×).

Biological Evaluation

The biocompatibility of the formulations was tested by subcutaneousinjections in 24 Sprague-Dawley rats. The animals were anaesthetized,shaved dorsally, and 1 mL of each formulation was injectedsubcutaneously. Two injections were performed on each side of themidline on the back of the rat. The animals were divided in two groups.One group was euthanized one week after injection, and the other groupthree weeks after injection. The animals were checked daily, and nosigns of illness were recorded. The injected formulations weremacroscopically and histologically well tolerated in all skin samples.Thus, no signs of necrosis or acute inflammation could be seen. Noformation of granulomas or signs of tissue reaction apart from a thinfibrous capsule (not shown) that surrounded the site of the implantedformulation were found. It could be concluded that the formulations werebiocompatible in rats.

Example 8 Hyaluronidase Degradation of Gels with Different Gel Strengths

A hyaluronate solution was prepared as set out in Example 1. Aftercomplete dissolution, the resulting hyaluronate solution had a pH above11.5, measured as described above. The hyaluronate solution was extrudedthrough an opening with a 0.5 mm diameter into a liquid medium of 99.5%ethanol. The extruded hyaluronate solution immediately precipitated intohyaluronate fibres.

Approximately 1 g of precipitated HA fibres was transferred to anaqueous/organic cross-linking medium containing 70% ethanol, BDDE andNaOH. The hyaluronate remained precipitated and in the form of fibres.The resulting cross-linking medium including HA, had a pH above 11.5.

The HA fibres were allowed to cross-link in the aqueous/organiccross-linking medium. The thus obtained cross-linked hyaluronate fibreswere then removed from the cross-linking medium and neutralized withphosphoric acid. The cross-linked HA fibres were dried in a vacuumchamber under reduced pressure (˜200 mbar) at room temperature forapproximately 2 hours.

The described precipitated intermediate of the cross-linked HA fibreswas allowed to swell to a defined HA concentration as follows.Phosphate-buffered saline solution was added to the dried HA fibres to aconcentration of about 20-40 mg HA/mL. The partly swollen gel was filledinto 10 mL plastic syringes. The filled syringes were sterilized usingmoist heat at 123° C. with a final F₀ of approximately 20 minutes. Thecontent of the autoclaved syringes was characterised regarding degree ofswelling (SwD procedure 2), HA concentration, minimum HA concentration(C_(min)), degree of modification (MoD), modification efficiency (MoE),and viscoelastic properties according to the methods described above.All the different products were subjected to hyaluronidase degradationaccording to “Determination of enzyme degradability” to verify that thebiodegradability of HA is maintained in the cross-linked product.

The results are reported in Tables 8A and 8B. The values of G′/(G′+G″)are >70% for all formulations which clearly shows that they all aregels. Furthermore, the G′ values show that the gels are firm.Nevertheless, the gels are degradable by hyaluronidase to more than 99%over a large span of gel strength, as shown by the determined swellingproperties and rheological properties. This shows that thebiodegradability of the native HA advantageously is maintained in thegel products according to the invention.

TABLE 8A Cross- linking process Characterisation Conc. Degradation bycross- SwD* C_(min) MoD hyaluronidase HA conc Sample linker (mL/g)(mg/mL) (%) (%) (mg/mL) 1 1x 75 14 0.2 >99 20 2 2x 26 39 0.4 >99 17 3**6x 17 59 1.9 >99 44 4 8x 17 59 1.6 >99 39 *Measured according toprocedure 2 **Cross-linking temperature 29° C.

TABLE 8B G′ G′ 1 Hz G″ 1 Hz G′ 0.1 Hz G″ 0.1 Hz G′ 1 Hz 0.1 Hz Sample(Pa) (Pa) (Pa) (Pa) (%)* (%)* 1 202 90 117 47 69% 71% 2 435 45 674 3691% 95% 3 7975 527 7207 689 94% 91% 4 6485 352 5914 478 95% 93% *Gelcharacteristic calculated as G′/(G′ + G″).

Example 9 Comparative Example

Five hyaluronic acid (HA) gel samples were prepared according to Example4 in EP 2 199 308 A1, see Table 9A below. A reference sample accordingto the invention (FU0509:1) was prepared using the cross-linkingprocedure set out in Example 1 hereinabove.

TABLE 9A Cross-linking conditions Stirring Temperature Reaction HAduring during time Sample supplier cross-linking cross-linking (h) 1Shiseido No RT 16 h 2 Shiseido Magnet RT 16 h 3 Shiseido No 45° C. 16 h4 Shiseido Magnet 40° C. 16 h 5 Food Propeller RT 16 h Chemifa ReferenceFood N/A RT 48 h sample Chemifa

TABLE 9B Results G′ G″ G′ G″ Sample MoD (%) 0.1 Hz 0.1 Hz 1 Hz 1 Hz 10.34 57 50 174 110 2 0.53 58 37 141 80 3 1.05 78 24 127 48 4 0.79 32 1358 28 5 1.70 145 6 157 10 Reference 0.36 853 40 932 53 sample

Gels were achieved for all samples, see Table 9B. The irregularly shapedgel particles provided by comparative samples 1-5 displayed MoD valuesin the same range as the sample prepared by the method according to theinvention. However, the G′ and G″ values are significantly lower for thecomparative samples 1-5 compared to the sample prepared by the methodaccording to the invention. It is concluded that the method according tothe invention is more efficient than the method disclosed in EP 2 199308 A1, since the present invention provides stronger gels already at alower MoD.

The microscopy images of comparative samples 1-5 (FIGS. 5A-E,respectively, each with 50x magnification) show a wide distribution inproduct size and shape, indicating that the method disclosed in EP 2 199308 A1 provides randomly sized particles. The shape and size of theresulting particles are not under control. In contrast, the microscopyimage of the product prepared by the method according to the invention(FIG. 5F) shows a product with a uniform size and shape.

Example 10 Ethanol Concentrations in the Precipitation Step

The effect of precipitating HA strings in different ethanolconcentrations was tested. Four aqueous ethanol solutions withconcentrations 65, 75, 85, 95% w/w were prepared. A solution of HA (5%w/w) in ethanol (35% w/w) with 0.5% NaOH (w/w) was extruded manually onsmall plastic (PE) films, which were immersed in baths with each of theethanol solutions and pure (99.5% w/w) ethanol, or stepwise immersed forone min in 75% and 85% ethanol and then transferred to 99.5% ethanol.

The resulting precipitated strings were collected and visually evaluatedusing light microscopy. The sensory feeling when handling the stringswas also evaluated. The strings were observed to have a smoother andmore even surface and be more curled, more brittle and showed morebundle structure the higher the ethanol concentration had been in theprecipitation bath.

Strings that were stepwise precipitated (75%→85%→99.5%) behaved like thestrings that were only precipitated in 75 w/w % or 85 w/w %. No completeprecipitation of the HA was observed with 65% ethanol under theseconditions. A sample that was kept in 65% ethanol for 20 min andthereafter fully precipitated in 99.5% ethanol had a completelydifferent appearance than the other strings; flat and thin band-likewithout bundle structures.

Example 11 Arranging a Desired Shape on a Hydrophobic Surface

The effect of the precipitation approach was tested by comparing the twofollowing procedures:

-   (I): 1) extrusion—2) placed on a PE film—3) precipitation in 99.5%    ethanol (c.f. Example 10)-   (II): 1) extrusion—2) free falling through 99.5% ethanol—3) landing    on a PE film

The resulting precipitated strings were collected and visually evaluatedusing light microscopy. The sensory feeling when handling the stringswas also evaluated. It was concluded that in the first type ofprecipitation procedure (I), smooth and even strings were achieved,while in the second type of precipitation procedure (II), brittle anduneven strings with bumps were obtained.

Example 12 Subcutaneous Injection in Rats

A gel formulation prepared in essentially the same way as described inExample 7 was tested in vivo by subcutaneous injection in hairless rats(Sprague Dawley). It was observed that the string shape of the implantedgel (FIG. 6A) remains after 6 months (FIG. 6B) in the subcutaneous areain the rat. FIG. 6A shows strings (non explant) diluted in Milli-RX andcoloured with Toluidine blue after extrusion through an 18 G cannula.FIG. 6B shows the same string batch explanted from subcutaneous area inrat after 6 months.

It is concluded that the string shape is retained after extrusionthrough a needle as well as after 6 months in rat.

Example 13 Comparative Example

US 2012/0034462 A1 suggests without experimental evidence that thinstrands of a cross-linked HA gel can be produced by passing a solid massof the cross-linked HA gel through a sieve or mesh. To test this theory,a gel was prepared according to Example 1 of US 2012/0034462 A1 (HA MW 3MDa; BDDE 75 mg/g HA; temperature 50° C. for 2 hours). The hydrogel waspassed through a 32 μm or 63 μm mesh screen once.

The resulting HA gel structures in 10× amplification are shown in FIG.7, (A) 32 μm mesh size; (B) 63 μm mesh size (bar=2 mm). The resultingparticles did not display any ordered structure, and in particular notany elongated shape.

The invention claimed is:
 1. Method for manufacturing a shapedcross-linked hyaluronic acid product, comprising the steps of: (i)providing a hyaluronic acid substrate dissolved in a first liquidmedium, which is an aqueous solution, without any cross-linking; (ii)precipitating the hyaluronic acid substrate by subjecting it to a secondliquid medium comprising an amount of one or more first water-solubleorganic solvent(s) giving precipitating conditions for hyaluronic acidwithout any cross-linking; wherein step (i) and/or step (ii) furthercomprises arranging the hyaluronic acid substrate in a desired shape;and (iii) subjecting the non-cross-linked precipitated hyaluronic acidsubstrate in the desired shape to a single cross-linking reaction in athird liquid medium having a pH of 11.5 or higher and comprising one ormore polyfunctional cross-linking agent(s) and an amount of one or moresecond organic solvent(s) giving precipitating conditions for hyaluronicacid, under suitable conditions to obtain a precipitated, shapedcross-linked hyaluronic acid product, wherein the aqueous solution ofstep (i) contains 40-100 vol % water and 0-60 vol % of lower alkylalcohol(s).
 2. Method according to claim 1, wherein the first two steps(i) and (ii) occur in the absence of a cross-linking agent, and whereinthe polyfunctional cross-linking agent(s) is added in the thirdcross-linking step (iii).
 3. Method according to claim 1, wherein step(i) further comprises arranging the hyaluronic acid substrate solutionin a desired shape on a hydrophobic surface; and wherein theprecipitation of the shaped hyaluronic acid substrate in step (ii)occurs on said hydrophobic surface.
 4. Method according to claim 1,wherein said shape is selected from the group consisting of a particle,a fibre, a string, a strand, a net, a film, a disc and a bead.
 5. Methodaccording to claim 4, wherein said shape is a fibre and the ratiobetween its length and its width is 10:1 or higher.
 6. Method accordingto claim 1, wherein step (ii) involves extruding the hyaluronic acidsubstrate into the second liquid medium comprising an amount of thefirst water-soluble organic solvent(s) giving precipitating conditionsfor hyaluronic acid, thereby allowing the extruded hyaluronic acidsubstrate to form a precipitated fibre in the second liquid medium. 7.Method according to claim 1, wherein the second liquid medium of step(ii) contains 0-30 vol % water and 70-100 vol % of the firstwater-soluble organic solvent(s).
 8. Method according to claim 1,wherein the third liquid medium of step (iii) contains 0-35 vol % water,65-100 vol % of the second organic solvent(s), and one or morepolyfunctional cross-linking agent(s).
 9. Method according to claim 1,wherein the organic solvent(s) is individually selected from one or morelower alkyl alcohol(s).
 10. Method according to claim 9, wherein thelower alkyl alcohol is ethanol.
 11. Method according to claim 1, saidpolyfunctional cross-linking agent(s) being individually selected fromthe group consisting of 1,4-butanediol diglycidyl ether (BDDE),1,2-ethanediol diglycidyl ether (EDDE) and diepoxyoctane.
 12. Methodaccording to claim 11, wherein said polyfunctional cross-linking agentis 1,4-butanediol diglycidyl ether (BDDE).
 13. Method according to claim1, further comprising the steps of: (iv) subjecting the precipitatedcross-linked hyaluronic acid product to non-precipitating conditions;and (v) isolating the cross-linked hyaluronic acid product innon-precipitated form.
 14. Method according to claim 13, wherein step(v) further comprises sterilizing the cross-linked hyaluronic acidproduct.